Plant pest control

ABSTRACT

The present invention is drawn to a novel class of proteins, and their receptors. Novel processes, assays and methods for controlling plant pests are provided.

This is a national stage application of PCT/EP/98/01952, filed Apr. 2,1998, and published Oct. 8, 1998, which is a continuation-in-part ofeach of the following U.S. patent applications Ser. No. 08/838,219,filed Apr. 3, 1997, now U.S. Pat. No. 5,877,012; Ser. No. 08/832,263,filed Apr. 3, 1997, abandoned; and Ser. No. 08/832,265, filed Apr. 3,1997, abandoned.

The present invention relates to a novel class of proteins for thecontrol of plant pests. Plant pests are a major factor in the loss ofthe world's commercially important agricultural crops resulting both ineconomic hardship to farmers and nutritional deprivation for localpopulations in many parts of the world. Broad spectrum chemicalpesticides have been used extensively to control or eradicate pests ofagricultural importance. There is, however, substantial interest indeveloping effective alternative pesticides.

Control of various pests through the use of biological molecules hasbeen possible in only a limited number of cases. The best known examplesof biological molecules with pesticidal uses are the δ-endotoxins fromBacillus thuringiensis (Bt), which is a gram-positive spore formingmicroorganism. Varieties of Bt are known that produce more than 25different but related δ-endotoxins. Bt strains produce δ-endotoxinsduring sporulation the use of which is limited because they are activeagainst only a very few of the many insect pests.

The limited specificity of the Bt endotoxins is dependent, at least inpart, on both the activation of the toxin in the insect gut (Haider, M.Z. et al., 1986, Eur. J. Biochem. 156:531-540) and its ability to bindto specific receptors present on the insect's midgut epithelial cells(Hofmann, C. P. et al., 1988, PNAS 85:7844-7848). Therefore, the abilityto control a specific insect pest using &endotoxins at present dependson the ability to find an appropriate δ-endotoxin with the desired rangeof activity. In many cases, no such δ-endotoxin is known, and it is notcertain that one even exists.

Plants also routinely become infected by fungi and bacteria, and manymicrobial species have evolved to utilize the different niches providedby the growing plant. In addition to infection by fungi and bacteria,many plant diseases are caused by nematodes which are soil-borne andinfect roots, typically causing serious damage when the same cropspecies is cultivated for successive years on the same area of ground.

The severity of the destructive process of disease depends on theaggressiveness of the phytopathogen and the response of the host, andone aim of most plant breeding programs is to increase the resistance ofhost plants to disease. Novel gene sources and combinations developedfor resistance to disease have typically only had a limited period ofsuccessful use in many crop-pathogen systems due to the rapid evolutionof phytopathogens to overcome resistance genes.

It is apparent, therefore, that scientists must constantly be in searchof new methods with which to protect crops against plant pests. It hasbeen found in the present invention a novel class of proteins which canbe used to control plant pests.

Programmed cell death is a process whereby developmental orenvironmental stimuli activate a genetic program that culminate in thedeath of the cell (Jacobson, M. D. et al., 1997, Cell 88: 347-354). Thisgenetic potential exists in most, if not all, multicellular organisms.In the case of invertebrates, programmed cell death appears to play adual role by being an integral part of both the insect developmentprocess and a response mechanism to infections particularly of viralnature (Clem, R. J. et al.,1991, Science 254: 1388-1390). Programmedcell death appears to be executed in several different manners leadingto either apoptosis, atrophy or differentiation. Apoptosis is one of thebest characterized types of programmed cell death encompassingcytological changes including membrane-bound apoptotic bodies andcytoplasmic blebbing as well as molecular changes such asendonucleolysis typified by the generation of oligosomal lengthfragments (Vaux, D. L and Strasser, A., 1996, PNAS 93:2239-2244).Although the overall apoptotic phenomenology is rather conserved amongthe different organisms, it is interesting to point out that, for manyinsect cells, cytoplasmic vacuolization and swelling rather thancondensation seem to be the cytological features associated withapoptotic processes (Bowen, I. D., et al.,1996, Micros. Res.Techniq.34:202-217). The novel class of proteins disclosed within thepresent invention are shown to induce programmed cell death and exert apesticidal effect.

The present invention is drawn to VIP3A(c) proteins including homologuesthereof. Also provided by the invention are domains of proteins of theVIP3 class, including the toxic domain and the stabilizing domain. Apreferred embodiment of the invention is the toxic domain of theVIP3A(a) protein and homologues thereof. Another preferred embodimentare antibodies to proteins of the VIP3 class, but preferably to theVIP3A(c) protein.

The invention also provides hybrid toxins comprising a toxic domain of aprotein of the VIP3 class. In a preferred embodiment, the hybrid toxinis a chimeric proteins having a toxic core domain operably linked to aheterologous stabilizing domain. In another preferred embodiment, thehybrid toxin comprises an antibody, or immunologically-active fragmentthereof, which immunologically recognizes the VIP3 receptor operablylinked to a toxic domain from other proteins, wherein the toxin domainis obtained from a number of cytotoxic proteins including but notlimited to Bacillus toxins, including endotoxins and vegetativeinsecticidal proteins.

Also encompassed by the invention are plants comprising a DNA sequencewhich encodes a protein of the VIP3 class, but preferably a VIP3A(c)protein. Preferred embodiments include plants selected from the groupconsisting of maize, sorghum, wheat, sunflower, tomato, cole crops,cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, andoilseed rape. In a particularly preferred embodiment, the plant is amaize plant.

The invention also provides microorganisms comprising a heterologous DNAsequence which encodes a protein of the VIP3 class, but preferably aVIP3A(c) protein. In a preferred embodiment, the microorganism isselected from the group consisting of bacteria, baculovirus, algae andfungi. In another preferred embodiment, the microorganism is selectedfrom the group consisting of Bacillus, Pseudomonas, Clavibacter, andRhizobium. Further encompassed by the invention are entomocidalcompositions comprising microorganisms comprising a heterologous DNAsequence which encodes a protein of the VIP3 class, but preferably aVIP3A(c) protein.

The invention further relates to plants and microorganisms furthercomprising a second DNA sequence which encodes a second insecticidalprotein. Particularly preferred second DNA sequences are those whichencode a δ-endotoxin, those which encode another protein of the VIP3class, or those which encode a protein of the VIP1 or VIP2 classes. In amore preferred embodiment, the δ-endotoxin is active against an insectselected from the group consisting of Lepidoptera and Coleoptera. In amore particularly preferred embodiment the δ-endotoxin is active againstOstrinia, or Diabrotica. In another particularly preferred is a secondDNA sequence which encodes a δ-endotoxin protein selected from the groupconsisting of Cry1, Cry3, Cry5 and Cry9. In a more particularlypreferred embodiment, the δ-endotoxin is selected from the groupconsisting of Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1Ea,Cry1Fa, Cry3A, Cry9A, Cry9C and Cry9B. Most particularly preferred areδ-endotoxins selected from the group consisting of Cry1Ab, Cry1Ba andCry9C proteins.

The invention further provides a method of controlling insects bycontacting the insects with an insecticidal amount of a protein of theVIP3 class, but preferably a VIP3A(c) protein, or an insecticidal amountof a chemical ligand to a receptor of the VIP3 class of proteins. In onepreferred embodiment, the insects are contacted with a transgenic plantcomprising a DNA sequence which expresses a protein of the VIP3 class,but preferably a VIP3A(c) protein in another preferred embodiment, theinsects are contacted with a an entomocidal composition comprising aprotein of the VIP3 class, but preferably a VIP3A(c) protein, orcomprising a DNA sequence which expresses a protein of the VIP3 class,but preferably a VIP3A(c) protein. In another preferred embodiment, thetransgenic plant comprises a DNA sequence which expresses the VIP3A(a)protein. In another preferred embodiment the insect is selected from thegroup consisting of Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, and Acari. In aparticularly preferred embodiment, the insect is a Coleoptera orLepidoptera. In another particularly preferred embodiment, the insect isselected from the group consisting of black cutworm (Agrotis ipsilon),fall armyworm (Spodoptera frugiperda), beet armyworm (S. exigua), yellowstriped armyworm (S. omithogalli), southwestern corn borer (Diatraeagrandiosella), sugarcane borer (D. saccharalis), corn earworm(Helicoverpa zea), mediterranean corn borer (Sesamia nonagroides),cabbage looper (Trichoplusia ni), velvetbean caterpillar (Anticarsiagemmatalis), diamondback moth (Plutella xylostella) and tobacco budworm(Heliothis virescens).

Also provided by the invention is a method of controlling insectswherein the transgenic plant or microorganism further comprises a secondDNA sequence which encodes a second insecticidal protein such as thosementioned hereinbefore.

The invention further provides recombinant DNA sequences which encode aVIP3A(c) protein including homologues thereof. In another preferredembodiment, the DNA sequence is a synthetic sequence which has beenaltered for optimum expression in a plant, particularly where the DNAsequence has been optimized for expression in a maize plant. Alsopreferred are DNA sequences which comprise both a synthetic portion anda native portion. In a particularly preferred embodiment, the DNAsequence encoding the VIP3A(c) protein has been optimized for expressionin a maize plant. Another preferred embodiment are DNA sequences whichare homologous to a DNA sequence which encodes a VIP3A(c) protein.Particularly preferred are DNA sequences which hybridize undermoderately stringent conditions to the vip3A(c) coding sequence. Yetanother embodiment of the invention is a recombinant DNA sequence whichexpresses a protein of the VIP3 class, preferably a VIP3A(c) protein,under the control of a heterologous promoter, or wherein the codingregions is incorporated into the genome of an organism where it is notnaturally expressed or is expressed at higher levels than that occurringnaturally.

The invention is further drawn to a method of identifying and isolatinghomologues of a VIP3A(c) protein. or of a DNA sequence which encodessaid protein.

Also provided by the invention are expression cassettes comprising apromoter operably linked to a DNA sequence encoding a protein of theVIP3 class, but preferably a VIP3A(c) protein. In one preferredembodiment the promoter is selected from the group consisting ofconstitutive, tissue-preferred and tissue-specific promoters forexpression in plants. In a particularly preferred embodiment, thepromoter is selected from the group consisting of the ubiquitin, PEPcarboxylase, LPT and MTL promoters. In another preferred embodiment, thepromoter is functional in a microorganism.

The invention further provides a receptor to a protein of the VIP3 classand DNA sequences which. In one embodiment of the invention, thereceptor comprises a death domain and a repeated EGF-motif. A morepreferred embodiment of the invention comprises a receptor to theVIP3A(a). A more particularly preferred embodiment is the receptorprotein sequence set forth in SEQ ID NO:9, and homologues thereto. Alsoencompassed by the invention are DNA sequences which encode thesereceptor proteins, e.g., the DNA sequence set forth in SEQ ID NO:8 andhomologues thereto. The cDNA for the VIP3 receptor is contained inplasmid pCIB7113, which was deposited under the Budapest Treaty with theNRRL [Agricultural Research Service, Patent Culture Collection (NRRL),Northern Regional Research Center, 1815 North University Street, Peoria,Ill. 61604, USA] on Mar. 29, 1997 and has accession number B-21676.Antibodies to a receptor of the VIP3 class of proteins are alsoencompassed by the invention.

Also provided by the invention is a method of identifying a compound asa VIP3 receptor chemical ligand having pesticidal activity comprisingexposing a cell, preferably an insect cell, to a test compound, andassaying said cell for apoptotic activity. In another embodiment of theinvention, the method comprises measuring specific binding between VIP3receptor and a test compound. A preferred embodiment are VIP3 receptorligands identified by the method.

Definitions

“Plant pest” means any organism known to associate with plants andwhich, as a result of that association, causes a detrimental effect onthe plant's health and vigor. Plant pests include but are not limited tofungi, bacteria, insects, and nematodes. The term plant as used hereinencompasses whole plants and parts of plants such as roots, stems,leaves and seed, as well as cells and tissues within the plants or plantparts.

The “VIP3 class of proteins” comprises VIP3A(a), VIP3A(b) VIP3A(c) andtheir homologues. “Homologue” is used throughout to mean that theindicated protein or polypeptide bears a defined relationship to othermembers of the VIP3 class of proteins. This defined relationshipincludes but is not limited to, 1) proteins which are at least 70%, morepreferably 80% and most preferably 90% identical at the sequence levelto another member of the VIP3 class of proteins while also retainingpesticidal activity, 2) proteins which are cross-reactive to antibodieswhich immunologically recognize another member of the VIP3 class ofproteins, 3) proteins which are cross-reactive with a receptor toanother member of the VIP3 class of proteins and retain the ability toinduce programmed cell death, and 4) proteins which are at least 70%,more preferably 80% and most preferably 90% identical at the sequencelevel to the toxic core region of another member of the VIP3 class ofproteins while also retaining pesticidal activity.

A “hybrid toxin” is used to indicate a genetic fusion, having domainsoperably linked so that, when translated, a functional chimeric proteinis formed having, in the aggregate, the properties of the individualdomains. “Domain” is used to indicate a region or portion of a proteinor confers a recognizable function or structure which contributes to theoverall functionality of the protein. It is recognized that a DNAsequence which encodes a protein domain is also encompassed by thisdefinition.

“Heterologous” is used to indicate that a protein, polypeptide ornucleotide sequence has a different natural origin with respect to itscurrent host. For example, if a vip3A(a) gene from a Bacillusthuringiensis is genetically transformed into a plant cell, then thegene is described as being heterologous with respect to its currenthost, which is the plant cell. Furthermore, if a vip3A(a) gene fromBacillus thuringiensis is genetically transformed into a Pseudomonasbacterium, then the gene is also described as being heterologous withrespect to the Pseudomonas. “Heterologous” is also used to indicate thatone or more of the domains present in a chimeric protein, polypeptide ornucleotide sequence differ in their natural origin with respect to otherdomains present. For example, if the toxic domain from VIP3A(a) proteinis fused to the binding domain from the VIP1 A(a) protein to make afunctional insecticidal protein, then the chimeric fusion would havedomains that are heterologous to each other. In addition, a heterologouschimeric protein or polypeptide comprising the fusion of a toxic domainfrom VIP3A(a) protein to the binding domain from the VIP1A(a) protein,when expressed in a plant, would also be considered heterologous withrespect to the plant host.

The term “chimeric” is used to indicate that the protein, polypeptide,or nucleotide sequence is comprised of domains at least one of which hasan origin that is heterologous with respect to the other domainspresent. These chimeric proteins or polypeptides are encoded by chimericnucleotide sequences which have been fused or ligated together resultingin a coding sequence which does not occur naturally. Such chimericconstructions may also be designated as “recombinant.”

“Expression cassette” as used herein means a DNA sequence capable ofdirecting expression of a gene in plant cells, comprising a promoteroperably linked to an amino acid coding region which is operably linkedto a termination region. The gene may be chimeric, meaning that at leastone component of the gene is heterologous with respect to at least oneother component of the gene. The gene may also be naturally occurring,but which has been obtained in a recombinant form useful for genetictransformation of a plant or microorganism.

Figures

FIG. 1: Amino acid sequence (SEQ ID NO: 9) of the receptor for VIP3A(a)translated from the cDNA of SEQ ID NO: 8. Several features of theprotein are shown: dotted line—signal peptide (amino acid 13 to 35);italic—domain spanning the putative death domain (amino acid 81-205);double underline—sequences with strong homology to sequences found inconsensus death domains; bold—CKC motif repeated six times spanning theEGF-motifs; underline—sequences repeated within the EGF-motifs.

Arthropod Pests

For purposes of the present invention, pests include insects andarachnids selected from the orders Coleoptera, Diptera, Hymenoptera,Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera,Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera,and Acari, particularly Coleoptera and Lepidoptera.

A list of pests associated with major crop plants are provided, forexample, in Tables 1-10 on pages 13 to 20 of WO 96/10083, which isincorporated herein by reference. Further pests are provided in thefollowing Tables 1-8. Such pests are included within the scope of thepresent invention.

TABLE 1 Lepidoptera (Butterflies and Moths) Maize Crucifers (broccoli,cabbage, Sesamia nonagroides, cauliflower, collards) Mediterranean cornborer Artogeia rapae, imported cabbageworm Ostrinia furnacalis, Asiancorn Pieris brassicae, cabbage butterfly borer Trichoplusia ni, cabbagelooper Cotton Plutella xylostella, diamondback moth Helicoverpaarmigera, cotton Spodoptera exigua, beet armyworm bollworm Agrotisipsilon, black cutworm Rice Agrotis segetum, common cutworm Chilosuppressalis, asiatic rice Mamestra configura, bertha army worm borerGrapes Scirpophaga sp. Endopiza viteana, grape berry moth TomatoDeciduous Fruits and Nuts Helicoverpa zea, tomato Cydia pomonella,codling moth fruitworm Platynota idaeusalis, tufted apple Spodopteraexigua, beet bud moth armyworm Peppers Spodoptera frugiperda, fallOstrinia nubilalis, European corn borer armyworm Spodoptera exigua, beetarmyworm Spodoptera ornithogalli, Spodoptera eridania, southernyellowstriped armyworm armyworm Spodoptera praefica, western Potatoyellowstriped armyworm Ostrinia nubilalis, European corn borerSpodoptera eridania, southern Phthorimaea operculella, potato armywormtuberworm Agrotis ipsilon, black cutworm Canola Peridroma saucia,variegated Plutella xylostella, diamondback moth cutworm SugarcanePapaipema nebris, stalk borer Diatraea saccharalis, sugarcane borerTrichoplusia ni, cabbage looper Keiferia lycopersicella, tomato pinwormManduca sexta, tobacco hornworm Manduca quinquemaculata, tomato hornworm

TABLE 1 Lepidoptera (Butterflies and Moths) Maize Crucifers (broccoli,cabbage, Sesamia nonagroides, cauliflower, collards) Mediterranean cornborer Artogeia rapae, imported cabbageworm Ostrinia furnacalis, Asiancorn Pieris brassicae, cabbage butterfly borer Trichoplusia ni, cabbagelooper Cotton Plutella xylostella, diamondback moth Helicoverpaarmigera, cotton Spodoptera exigua, beet armyworm bollworm Agrotisipsilon, black cutworm Rice Agrotis segetum, common cutworm Chilosuppressalis, asiatic rice Mamestra configura, bertha army worm borerGrapes Scirpophaga sp. Endopiza viteana, grape berry moth TomatoDeciduous Fruits and Nuts Helicoverpa zea, tomato Cydia pomonella,codling moth fruitworm Platynota idaeusalis, tufted apple Spodopteraexigua, beet bud moth armyworm Peppers Spodoptera frugiperda, fallOstrinia nubilalis, European corn borer armyworm Spodoptera exigua, beetarmyworm Spodoptera ornithogalli, Spodoptera eridania, southernyellowstriped armyworm armyworm Spodoptera praefica, western Potatoyellowstriped armyworm Ostrinia nubilalis, European corn borerSpodoptera eridania, southern Phthorimaea operculella, potato armywormtuberworm Agrotis ipsilon, black cutworm Canola Peridroma saucia,variegated Plutella xylostella, diamondback moth cutworm SugarcanePapaipema nebris, stalk borer Diatraea saccharalis, sugarcane borerTrichoplusia ni, cabbage looper Keiferia lycopersicella, tomato pinwormManduca sexta, tobacco hornworm Manduca quinquemaculata, tomato hornworm

TABLE 3 Homoptera (Whiteflies, Aphids etc. . .) Rice Melon Nilaparvatalugens Bemisia argentifolii, silverleaf Sogatella furcifera whiteflyLaodelphaax striatellus Bemisia tabaci, sweetpotato Tomato whiteflyMyzus persicae, green peach aphid Carrot Macrosiphum euphorbiae, potatoaphid Cavariella aegopodii, carrot Trileurodes vaporariorum, greenhouseaphid whitefly Canola Bemisia tabaci, sweetpotato whitefly Brevicorynebrassicae, cabbage Bemisia argentifolii, silverleaf whitefly aphidCrucifers (broccoli, cabbage, Vegetables cauliflower, collards) Aphisfabae, bean aphid Brevicoryne brassicae, cabbage aphid Sugar Beet Myzuspersicae, green peach aphid Pemphigus popullivenae, sugar Peppers beetroot aphid Myzus persicae, green peach aphid Deciduous Fruits and NutsPotato Dysaphis plantaginea, rosy Empoasca fabae, potato leafhopperapple aphid Myzus persicae, green peach aphid Sugarcane Macrosiphumeuphorbiae, potato aphid Saccharosydne saccharivora, Paratriozacockerelli, potato psyllid West Indian canefly Sipha flava, yellowsugarcane aphid

TABLE 3 Homoptera (Whiteflies, Aphids etc. . .) Rice Melon Nilaparvatalugens Bemisia argentifolii, silverleaf Sogatella furcifera whiteflyLaodelphaax striatellus Bemisia tabaci, sweetpotato Tomato whiteflyMyzus persicae, green peach aphid Carrot Macrosiphum euphorbiae, potatoaphid Cavariella aegopodii, carrot Trileurodes vaporariorum, greenhouseaphid whitefly Canola Bemisia tabaci, sweetpotato whitefly Brevicorynebrassicae, cabbage Bemisia argentifolii, silverleaf whitefly aphidCrucifers (broccoli, cabbage, Vegetables cauliflower, collards) Aphisfabae, bean aphid Brevicoryne brassicae, cabbage aphid Sugar Beet Myzuspersicae, green peach aphid Pemphigus popullivenae, sugar Peppers beetroot aphid Myzus persicae, green peach aphid Deciduous Fruits and NutsPotato Dysaphis plantaginea, rosy Empoasca fabae, potato leafhopperapple aphid Myzus persicae, green peach aphid Sugarcane Macrosiphumeuphorbiae, potato aphid Saccharosydne saccharivora, Paratriozacockerelli, potato psyllid West Indian canefly Sipha flava, yellowsugarcane aphid

TABLE 3 Homoptera (Whiteflies, Aphids etc. . .) Rice Melon Nilaparvatalugens Bemisia argentifolii, silverleaf Sogatella furcifera whiteflyLaodelphaax striatellus Bemisia tabaci, sweetpotato Tomato whiteflyMyzus persicae, green peach aphid Carrot Macrosiphum euphorbiae, potatoaphid Cavariella aegopodii, carrot Trileurodes vaporariorum, greenhouseaphid whitefly Canola Bemisia tabaci, sweetpotato whitefly Brevicorynebrassicae, cabbage Bemisia argentifolii, silverleaf whitefly aphidCrucifers (broccoli, cabbage, Vegetables cauliflower, collards) Aphisfabae, bean aphid Brevicoryne brassicae, cabbage aphid Sugar Beet Myzuspersicae, green peach aphid Pemphigus popullivenae, sugar Peppers beetroot aphid Myzus persicae, green peach aphid Deciduous Fruits and NutsPotato Dysaphis plantaginea, rosy Empoasca fabae, potato leafhopperapple aphid Myzus persicae, green peach aphid Sugarcane Macrosiphumeuphorbiae, potato aphid Saccharosydne saccharivora, Paratriozacockerelli, potato psyllid West Indian canefly Sipha flava, yellowsugarcane aphid

TABLE 6 Diptera (Flies and Mosquitoes) Tomato Carrot Liriomyza trifolii,leafminer Psilia rosae, carrot rust fly Liriomyza sativae, vegetableleafminer Sugarbeet Scrobipalpula absoluta, tomato leafminer Tetanopsmyopaeformis, Crucifers (broccoli, cabbage, cauliflower, sugarbeet rootmaggot collards) Vegetables Delia brassicae, cabbage maggot Liviomyzasativae, vegetable Delia radicum, cabbage root fly leaf miner

TABLE 6 Diptera (Flies and Mosquitoes) Tomato Carrot Liriomyza trifolii,leafminer Psilia rosae, carrot rust fly Liriomyza sativae, vegetableleafminer Sugarbeet Scrobipalpula absoluta, tomato leafminer Tetanopsmyopaeformis, Crucifers (broccoli, cabbage, cauliflower, sugarbeet rootmaggot collards) Vegetables Delia brassicae, cabbage maggot Liviomyzasativae, vegetable Delia radicum, cabbage root fly leaf miner

TABLE 6 Diptera (Flies and Mosquitoes) Tomato Carrot Liriomyza trifolii,leafminer Psilia rosae, carrot rust fly Liriomyza sativae, vegetableleafminer Sugarbeet Scrobipalpula absoluta, tomato leafminer Tetanopsmyopaeformis, Crucifers (broccoli, cabbage, cauliflower, sugarbeet rootmaggot collards) Vegetables Delia brassicae, cabbage maggot Liviomyzasativae, vegetable Delia radicum, cabbage root fly leaf miner

For purposes of the present invention, pests also include fungalphytopathogens of plants. A list of fungal pests associated with majorcrop plants is provided in Table 9. Such pests are included within thescope of the present invention.

TABLE 9 Fungal Diseases of Plants Ear Molds Gibberella ear moldGibberella zeae G. saubinetti Aspergillus ear rot Aspergillus flavus A.parasiticus Diplodia ear rot Diplodia maydis D. macrospora Fusarium earrot Fusarium moniliforme F. monilif. var. subglutinans Stalk RotsPythium stalk rot Pythium aphanidermata Anthracnose stalk rotColletotrichum graminicola C. tucumanensis Glomerella graminicolaDiplodia stalk rot Diplodia maydis D. zeae-maydis Stenocarpella maydisMacrodiplodia zeae Sphaeria maydis S. zeae D. macrospora Fusarium stalkrot Fusarium moniliforme Gibberella stalk rot G. zeae G. saubinettiStewart's wilt & leaf blight Erwinia stewartii Leaf Diseases Northerncorn leaf blight Exserohilum turcicum Southern corn leaf blightBipolaris maydis Gray leaf spot Cercospora zeae-maydis C. sorghi var.maydis Anthracnose leaf blight Colletotrichum graminicola Common rustPuccinia sorghi P. maydis Southern rust Puccinia polysora Dicaeomapolysorum Head smut Sphacelotheca reiliana Common smut Ustilago maydisCarbonum leaf spot Helminthosporium carbonum Eye spot Kabatiella zeaeDowny Mildews Sorghum downy mildew Peronosclerospora sorghi Brown stripedowny mildew Sclerophthora rayssiae Sugarcane downy mildewPeronosclerospora sacchari Phillipine downy mildew Peronoscler.philippinensis Java downy mildew Peronosclerospora maydis Spontaneumdowny mildew Peronosclerospora spantanea Rajasthan downy mildewPeronosclerospora heteropogoni Graminicola downy mildew Sclerosporagraminicola Rusts Puccinia graminis f.sp. tritici Puccinia reconditaf.sp. tritici Puccinia striiformis Smuts Tilletia tritici Tilletiacontroversa Tilletia indica Ustilago tritici Urocystis tritici Rootrots, Foot rots and Blights Gaeumannomyces graminis Pythium spp.Fusarium culmorum Fusarium graminaerum Fusarium avenaceum Drechsleretritici-repentis Rhizoctonia spp. Colletotrichum graminicolaHelminthosporium spp. Microdochium nivale Pseudocercosporellaherpotrichoides Mildews Erysiphe graminis f.sp. tritici Sclerophthoramacrospora Miscellaneous Fungal Diseases Septoria tritici Septorianodorum

The proteins of the VIP3 class are secreted to the media by Bacillusspp. in vegetative stages of growth. VIP3A(a) is a member of a newlydiscovered class of proteins displaying insecticidal activity against abroad spectrum of lepidopteran insects including black cutworm (Agrotisipsilon), fall armyworm (Spodoptera frugiperda), beet armyworm (S.exigua), yellow striped armyworm (S. ornithogalli), southwestern cornborer (Diatraea grandiosella), sugarcane borer (D. saccharalis), cornearworm (Helicoverpa zea), Mediterranean corn borer (Sesamianonagroides), cabbage looper (Trichoplusia ni), velvetbean caterpillar(Anticarsia gemmatalis), diamondback moth (Plutella xylostella) andtobacco budworm (Heliothis virescens). Some of these lepidopteraninsects have been shown to be very resistant to other insecticidalproteins such as δ-endotoxin. For example, the reported LC₅₀ forCry1A(c), which is one of the most effective δ-endotoxin against blackcutworm, is greater than 6000 ng/cm² (Macintosh et al., J. lnvertebr.Pathol. 56:258-266 (1990)). In contrast, it takes 260-fold less ofVIP3A(a) protein to kill 50% of the black cutworm larvae. Thus, theVIP3A(a) protein displays a unique spectrum of insecticidal activities.

The present invention provides a new member of the VIP3 class ofproteins, the VIP3A(c) protein isolated from strain AB51 (pCIB7112deposited on Mar. 28, 1997 as Accession No. NRRL B-21675; all depositswere made in accordance with the Budapest Treaty by submission to theAgricultural Research Service, Patent Culture Collection (NRRL),Northern Regional Research Center, 1815 North University Street, Peoria,Ill. 61604, USA) as disclosed in SEQ ID NO:5-6.

The VIP3A(c) protein encoding DNA sequence was identified and isolated sthrough the use of PCR technology. In particular, primer sequences canbe made which recognize either conserved or variable regions of thecoding sequence, and are then used to screen DNA samples obtained fromeither known or unknown strains. It is recognized that there aremultiple approaches to identifying and isolating homologues within theVIP3 class of proteins and the DNA sequences which encode them, whichapproaches are well known to those skilled in the art.

The DNA and protein sequences for the VIP3A(a) and VIP3A(c) proteins arealigned on Table 10.

TABLE 10 Alignment of VIP3A(a) (Upper Line) against VIP3A(c) (LowerLine) 1 MNKNNTKLSTRALPSFIDYFNGIYGFATGIKDTMNMIFKTDTGGDLTLDE 50 SEQ IDNO:2 |||||.||||||||||||||||||||||||||||||||||||||||.||| 1MNKNNAKLSTRALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGDLALDE 50 SEQ ID NO:6 51ILKNQQLLNDISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVL 100||.||||||||||||||||||||||||||||||||||||||||||||||| 51ILENQQLLNDISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVL 100 101NDVNNKLDAINTMLRVYLPKITSMLSDVMKQNYALSLQIEYLSKQLQEIS 150|||||||||||||||||||||||||||||||||||||||||||||||||| 101NDVNNKLDAINTMLRVYLPKITSMLSDVMKQNYALSLQIEYLSKQLQEIS 150 151DKLDIINVNVLINSTLTEITPAYQRIKYVNEKFEELTFATETSSKVKKDG 200|||||||||||||||||||||||||||||||||||||||||||||||||| 151DKLDIINVNVLINSTLTEITPAYQRIKYVNEKFEELTFATETSSKVKKDG 200 201SPADILDELTELTELAKSVTKNDVDGFEFYLNTFHDVMVGNNLFGRSALK 250||||| |||.||||||||||.||||||||||||||||||||||||||||| 201SPADIRDELSELTELAKSVTQNDVDGFEFYLNTFHDVMVGNNLFGRSALK 250 251TASELITKENVKTSGSEVGNVYNFLIVLTALQAQAFLTLTTCRKLLGLAD 300||||||||||||||||||||||||||||||||||||||||.||||||||| 251TASELITKENVKTSGSEVGNVYNFLIVLTALQAQAFLTLTPCRKLLGLAD 300 301IDYTSIMNEHLNKEKEEFRVNILPTLSNTFSNPNYAKVKGSDEDAKMIVE 350|||||||||||||||||||||||||||||||||||||||||||||||||| 301IDYTSIMNEHLNKEKEEFRVNILPTLSNTFSNPNYAKVKGSDEDAKMIVE 350 351AKPGHALIGFEISNDSITVLKVYEAKLKQNYQVDKDSLSEVIYGDMDKLL 400|||||||||||||||||||||||||||||||||||||||||||||||||| 351AKPGHALIGFEISNDSITVLKVYEAKLKQNYQVDKDSLSEVIYGDMDKLL 400 401CPDQSEQTYYTNNIVFPNEYVITKIDFTKKMKTLRYEVTANFYDSSTGEI 450|||||:|||||||||||||||||||||||||||||||||||||||||||| 401CPDQSGQIYYTNNIVFPNEYVITKIDFTKKMKTLRYEVTANFYDSSTGEI 450 451DLNKKKVESSEAEYRTLSANDDGVYMPLGVISETFLTPINGFGLQADENS 500|||||||||||||||||||||||||||||||||||||||||||||||||| 451DLNKKKVESSEAEYRTLSANDDGVYMPLGVISETFLTPINGFGLQADENS 500 501RLITLTCKSYLRELLLATDLSNKETKLIVPPSGFISNIVENGSIEEDNLE 550|||||||||||||||||||||||||||||||||||||||||||||||||| 501RLITLTCKSYLRELLLATDLSNKETKLIVPPSGFISNIVENGSIEEDNLE 550 551PWKANNKNAYVDHTGGVNGTKALYVHKDGGISQFIGDKLKPKTEYVIQYT 600|||||||||||||||||||||||||||||||||||||||||||||||||| 551PWKANNKNAYVDHTGGVNGTKALYVHKDGGISQFIGDKLKPKTEYVIQYT 600 601VKGKPSIHLKDENTGYIHYEDTNNNLEDYQTINKRFTTGTDLKGVYLILK 650|||||||||||||||||||||||||||||||||||||||||||||||||| 601VKGKPSIHLKDENTGYIHYEDTNNNLEDYQTINKRFTTGTDLKGVYLILK 650 651SQNGDEAWGDNFIILEISPSEKLLSPELINTNNWTSTGSTNISGNTLTLY 700|||||||||||||||||||||||||||||||||||||||||||||||||| 651SQNGDEAWGDNFIILEISPSEKLLSPELINTNNWTSTGSTNISGNTLTLY 700 701QGGRGILKQNLQLDSFSTYRVYFSVSGDANVRIRNSREVLFEKRYM 746|||||||||||||||||||||||||||||||||||||||||||||||: : 701QGGRGILKQNLQLDSFSTYRVYFSVSGDANVRIRNSREVLFEKKDI 746

Polypeptide Domains of the VIP3 Class of Proteins

It has been shown that the VIP3A(a) protein undergoes proteolyticprocessing when mixed with the gut fluids of insect larvae. When gutfluids isolated from black cutworm are mixed with purified VIP3A(a),four major proteolytic products derived from VIP3A(a) can be identifiedhaving a molecular weight of approximately 66, 45, 33 and 22 kDa. The 22kDa band comprises the N-terminal portion of the VIP3A(a) protein fromamino acid 1 to amino acid 198 of SEQ ID NO:2. The 66 kDa band comprisesthe rest of the VIP3A(a) protein from amino acid 200 to amino acid 789of SEQ ID NO:2. Both the 45 and 33 kDa bands are derived by proteolysisfrom the 66 kDa band and constitute amino acid 412 to amino acid 789,and from amino acid 200 to amino acid 455, respectively, of SEQ ID NO:2.The 33 kDa band is the main component of the VIP3A(a) protein thatremains after an incubation period of more than two hours. This 33 kDa“toxic core” domain (amino acids 200 to 455 of SEQ ID NO:2) of theVIP3A(a) protein retains full insecticidal properties against a broadspectrum of lepidopteran insects. Similar results are obtained whenVIP3A(a) is incubated with gut fluids isolated from fall armyworm,another insect sensitive to VIP3A(a).

In addition to the toxic core domain, the VIP3A(a) protein alsopossesses a stabilizing domain at the C-terminus. The role of thestabilizing domain was explored using mutants of the VIP3A(a) proteinand the VIP3A(c) protein, neither of which display insecticidalproperties when ingested by insects known to be sensitive to VIP3A(a).When similar studies addressing the stability in black cutworm gut fluidwas conducted with VIP3A(a)-mutants, in particular with a mutant of theVIP3A(a) protein that contains three point mutations located at thecarboxy-terminal domain (amino acid 742 (E→D); amino acid 770 (S→P); andamino acid 784 (Y→H)), it was found that the protein was completelyhydrolyzed. Similar results were obtained for the VIP3A(c) (SEQ ID NO:6)protein isolated from AB51, which shares an overall identity of 96% withthe VIP3A(a) protein but lacks the carboxy-terminal domain of VIP3A(a).Both the mutant and VIP3A(c) protein, however, are active against theinsect cell line Sf-9. These results indicate that the function of thecarboxy-terminal domain of proteins of the VIP3 class is to providestability to the protein in the gut environment of susceptible insects.

Hybrid Toxins Comprising a VIP3 Region and a Heterologous Region

Toxins, enzymes, transcription factors, antibodies, cell bindingmoieties or other protein domains can be operably linked to the novelproteins of the present invention by producing in frame genetic fusionswhich, when translated by ribosomes, would produce a fusion protein withthe combined attributes of the VIP and the other component used in thefusion. Furthermore, if the protein domain fused to the VIP has anaffinity for another protein, nucleic acid, carbohydrate, lipid, orother chemical or factor, then a three-component complex can be formed.This complex will have the attributes of all of its components. Asimilar rationale can be used for producing four or more componentcomplexes. These complexes are useful as insecticidal toxins,pharmaceuticals, laboratory reagents, and diagnostic reagents, etc.Examples where such complexes are currently used are fusion toxins forpotential cancer therapies, reagents in ELISA assays and immunoblotanalysis.

The hybrid toxins of the invention include chimeric proteins having atoxic core domain which is heterologous to the stabilizing domain.Hybrid toxins are also created by combining an antibody, orimmunologically-active fragment thereof, which immunologicallyrecognizes the VIP3 receptor with a toxic domain from other proteins.The toxin domain is obtained from a number of cytotoxic proteins. Theseinclude but are not limited to Bacillus toxins, including endotoxins andvegetative insecticidal proteins. See for example U.S. application Ser.No. 08/037,057, filed Mar. 25, 1993 and U.S. application Ser. No.07/951,715 filed Sep. 25, 1992, herein incorporated by reference. Othertoxins include catalytic ribosome inactivators such as gelonin,Pseudomonas exotoxin A or phytolaccin, (the structure of Pseudomonasexotoxin has been well characterized in Chaudhary et al., J. Biol. Chem.265:16303-16310 (1990)); cell metabolism disrupters, such asribonucleases, (see, for example, Mariani et al. Nature 347:737-741(1990)); Barnase toxin (or PE-Bar), a chimeric toxin derived fromPseudomonas exotoxin A and a ribonuclease, (see, Prior et al. Cell64:1017-1023 (1991)); hydrophilic peptides that create pores inmembranes (see, Frohlich and Wells, Int. J. Peptide Protein Res. 37:2-6(1991)).

Mode of Action of VIP3A(a)

The VIP3A(a) protein has been shown to be active against a broadspectrum of plant pests. For example, histopathological observationsindicate that VIP3A(a) ingestion by susceptible insects such as blackcutworm (Agrotis epsilon) and fall armyworm (Spodoptera frugiperda)causes gut paralysis at concentrations as low as 4 ng/cm² of diet, withcomplete lysis of the gut epithelial cells resulting in larval death atconcentrations above 40 ng/cm². Less susceptible insects like Europeancorn borer (Ostrinia nubilalis) do not develop any pathology uponingesting VIP3A(a). While the proteolytic processing of the VIP3A(a)protein by midgut fluids obtained from susceptible and non-susceptibleinsects is comparable, in vivo immuno-localization studies show thatVIP3A(a) binding is restricted to gut cells of susceptible insects.Therefore, the insect host range for VIP3A(a) seems to be determined byits binding ability to gut cells. Histopathological observationsindicate that midgut epithelial cells of susceptible insects are theprimary target for the VIP3A(a) insecticidal protein and theirsubsequent lysis is the primary mechanism of lethality.

Programmed cell death is an active process of self-destruction thatseems to be important for development and maintenance of multicellularorganisms (Clem, R. J. et al Science 254: 1388-1390 (1991)). Cellsundergoing apoptosis, which is a form of programmed cell death, generatemembrane-bound apoptotic bodies and activate endogenous nucleases thatcleaves the chromatin into discrete fragments. SF-9 insect cells derivedfrom S. frugiperda exposed to the VIP3A(a) protein undergo a series ofcytological and molecular changes including membrane protrusions,profuse vacuolization and endonucleolysis which are indicative of anapoptotic-type of programmed cell death. Histological studies have shownthat the VIP3A(a) protein targets midgut epithelial cells of susceptibleinsects initiating a series of cytological changes comprising profusevacuolization and swelling prior to cell lysis and larval death. Thesemidgut cells also experienced an endonucleolysis process when exposed tothe VIP3A(a) protein as revealed by in situ detection of DNAfragmentation. These results indicate that VIP3A(a) exerts itsinsecticidal properties on susceptible insect cells by triggering anapoptotic-type of programmed cell death.

The Receptor for VIP3A(a) has been Isolated

The immunohistochemistry results provided above indicate that VIP3A(a)has the ability to bind to the apical membranes of midgut epithelialcells and that this binding triggers the process that will eventuallyend with cell lysis. This indicates that there exists one or moreproteins located in the apical membrane that recognize and bind toVIP3A(a) acting as a receptor. This receptor signals the interactionwith VIP3A(a) and triggers the process of apoptosis. Thus, the receptorwill mediate the response of the insect cell to VIP3A(a).

To isolate this receptor, a cDNA library was screened which was madefrom mRNA isolated from midgut tissue of black cutworm. The objective ofthe screen was to identify and isolate cDNA sequences which encodeproteins that will interact with VIP3A(a) in the two hybrid system (seeFields, S. and Song, O.-K. Nature 340:245-246 (1989)). This approachresulted in the identification and isolation of one cDNA whose encodedprotein strongly interacted with the VIP3A(a) protein. This 1.75 Kb-longcDNA (SEQ ID NO:8) encodes a protein of approximately 48 kDa (396 aminoacids; see SEQ ID NO:9). The cloned cDNA is similar in size to the mRNAencoding the cDNA as analyzed by Northern. A portion of the DNA sequencewhich encodes the first 5 to 20 amino acids may be missing. Thefollowing features can be identified in the cDNA encoded protein (seeFIG. 1): 1) it contains a signal peptide; 2) it contains a domain withhomology to the so-called death domain (Feinstein, E. et al. Trends inBiochem. 20:342-344 (1995)); and 3) it contains EGF-like motifs orrepeats (Fantl, W. J. et al. Annu. Rev. Biochem. 62:453-481 (1993)). Asearch of protein databases using the receptor of VIP3A(a) showedhomology with a family of extracellular glycoproteins known as Tenascins(Pearson, C. A. et al. EMBO J. 7:2677-2681 (1988)) or Hexabrachion(Nies, D. E. et al. J. Biol. Chem. 266:2818-2823 (1991)). This family ofproteins contains EGF-like repeats, interacts with multiple ligands, andperforms a role in cell adhesion and/or signaling. The combination of adeath domain and repeated EGF-motifs as observed in the VIP3 receptor isunique among programmed cell death receptors.

In addition, a portion of the VIP3A(a) receptor shares homology with theso-called “death domain.” The death domain is a 60 to 70 amino acid longmotif which is involved in protein to protein interaction and is sharedby proteins with diverse cellular functions (Feinstein, E. et al. Trendsin Biochem. 20:342-344 (1995)). Some of the protein members containingdeath domain motifs include receptors known to be associated withapoptotic processes. Some examples include the Fas receptor (Brakebush,C. et al. EMBO J. 11:943-950 (1992)) and the tumor necrosis factor (TNF)(Tartaglia, L. A. et al. Cell 74:845-853 (1993)).

Homologues to the VIP3A(a) receptor can be identified and isolated byvarious means, for example, by nucleic acid hybridization. Southern blotanalysis can be performed on DNA samples taken from insect cells orfungal cells that has been enzyme restricted, run in agarose and blottedonto nitrocellulose and/or nylon filters. The Southern blot can beprobed with the full-or partial length of the nucleic acid encoding thereceptor of the VIP3A(a) protein under low stringency hybridization andwashing conditions. The genes can be readily cloned and sequenced from acDNA or genomic library. A size-selected genomic library can also beobtained to facilitate cloning of the gene(s) of interests. Thetechnical protocols to perform the experiments outlined above arereadily available (see for instance Molecular Cloning, A LaboratoryManual, Second Edition, Vols. 1-3, Sambrook et al. (eds.) Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and referencetherein).

Antibodies to VIP3A(a) and its Receptor

Polyclonal and monoclonal antibodies to a VIP3 protein or its receptor,including fragments thereof which immunologically recognize a portion ofeither protein, are provided. The antibody and monoclonal antibodies ofthe present invention can be prepared by utilizing a VIP3 protein or itsreceptor as the antigen.

The antibodies of the invention include polyclonal and monoclonalantibodies as well as fragments thereof which retain their ability tobind a VIP3 protein or its receptor. An antibody, monoclonal antibody,or fragment thereof is said to be capable of binding a molecule if it iscapable of specifically reacting with the molecule to thereby bind themolecule to the antibody, monoclonal antibody, or fragment thereof. Theterm “antibody” (Ab) or “monoclonal antibody” (Mab) is meant to includeintact molecules as well as fragments or binding regions or domainsthereof (such as, for example, Fab and F(ab)₂ fragments) which arecapable of binding hapten. Such fragments are typically produced byproteolytic cleavage, such as papain or pepsin. Alternatively,hapten-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry.

Methods for the preparation of the antibodies of the present inventionare generally known in the art. For example, see Antibodies, ALaboratory Manual, Ed Harlow and David Lane (eds.) Cold Spring HarborLaboratory, NY (1988), as well as the references cited therein. Standardreference works setting forth the general principles of immunologyinclude: Klein, J. Immunology: The Science of Cell-NoncellDiscrimination, John Wiley & Sons, NY (1982); Dennett, R., et alMonoclonal Antibodies, Mybridoma: A New Dimension in BiologicalAnalyses, Plenum Press, NY (1980); and Campbell, A. “Monoclonal AntibodyTechnology,” In Laboratory Techniques in Biochemistry and MolecularBiology, Vol.13, Burdon et al. (eds.), Elsevier, Amsterdam (1984). Seealso, U.S. Pat. Nos: 4,609,893; 4,713,325; 4,714,681; 4,716,111;4,716,117; and 4,720,459.

It is recognized that following the methods described herein, antibodiesspecific for a particular VIP3 protein or its receptor can be generated.The subset of MAb lines which possess the desired binding specificitycan be used as a source of messenger RNA for cloning of the cDNA for theparticular monoclonal antibody.

The cloned DNA can then be sequenced by methods known in the art. See,for example, Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd. Edition, Cold Spring Harbor Laboratory Press, NY (1989) vol. 1-3,and the references cited therein. From the nucleic acid sequence, theprotein sequence of the binding region from the selected MAb can bededuced.

One use of the antibodies and monoclonal antibodies of the inventionincludes but is not limited to the production of hybrid toxin molecules.That is, when linked, the monoclonal antibody or antibody fragmentretains its binding properties and the toxin moiety retains itscytotoxic properties.

Various methods are known for obtaining antibody genes. One method is toclone a random library of antibody genes in a phage and screen thelibrary for ability to bind to a VIP3 protein or its receptor. Anotheravailable approach is to generate monoclonal antibodies which bind to aVIP3 protein or its receptor and then clone the antibody genes from suchlines. For the present example, the second method is used. Antibodygenes can be cloned from hybridoma cells using primers to conserved DNAsequences within the constant regions and the framework regions of thevariable regions and amplified for cloning using the polymerase chainreaction (PCR). See generally, Mullis et al., Meth. Enzymol.,155:335-350 (1987); Erlich, (ed.), PCR Technology, Stockton Press (N.Y.1989). A database of mouse heavy chain and light chain sequencescompiled by Kabat et al., U.S. Dept Health and Human Services, U.S.Government Printing Offices (1991) has been successfully used togenerate both isotype specific and degenerate primers for cloningantibody genes. (Jones et al. Bio/technology 9:88-89 (1991)).Additionally, techniques are well known for cloning of smaller fragmentsof antibodies (Fab) which possess the binding properties of the originalantibody. Complete antibodies are large molecules (150 kDa), but muchsmaller Fab and Fv antigen-binding fragments (12 kDa-50 kDa) have beenshown to retain full binding affinity. Single chain Fv fragments (scFv)in which Vh and VI domains are linked by a hydrophilic and flexiblepeptide have been used successfully to target enzymes and toxins tospecific cells (Bird, Science 423:423-426 (1988); Huston, PNAS85:5879-5883 (1988)). Single Vh domains (Dabs) and single complementarydetermining regions as small as 20 amino acids in length, called minimalrecognition units (m.r.u.), have also been used for antigen binding(Ward, Nature 341:544-546 (1989); Taub, J. Biol. Chem 264:259-265(1989); Williams, PNAS 86:5537-5541 (1989)). Thus, it is possible toreduce the binding domain specific for a VIP3 or its receptor to a verysmall size.

Polymerase chain reaction technology and specific oligonucleotideprimers are used to clone immunoglobulin genes or regions fromimmunoglobin genes. PCR primers specific for both the heavy and lightchains of IgM and the three IgG isotypes were selected from the Kabatdatabase described above. Primers for the region encoding theNH₂-terminal end of the mature variable region were designed to initiateat the first framework region and were made with some degeneracy toallow these to be used as “universal primers”. The 3′ primers used forthe specific PCR amplification of the variable regions were designedfrom conserved sequences of the first constant domain (CH1) of both thelight and heavy chains. A different 3′ primer is used for immunoglobulinisotypes IgG1, IgG3, and IgM. Isotypes IgG2A and IgG2B can be amplifiedwith the same primers used for IgG1. Antibody variable regions arecloned into a light and heavy chain expression vector containing anendoplasmic reticulum signal peptide and the constant regions of IgG1light and heavy chains, respectively.

Primer sequences used for the PCR cloning of the mouse immunoglobulinlight and heavy variable regions are available in the publishedliterature (Coloma et al. Bio/Techniques 11: 152-156 (1991); Jones etal. Bio/Technology 9:88-89 (1991)). Oligonucleotides were made on anApplied Biosystems DNA synthesizer 380B (Applied Biosystems, FosterCity, Calif.) using standard conditions as described below. The PCRprimers incorporate restriction sites and, after amplification anddigestion, can be cloned into a plant expression vector under thecontrol of a plant-expressible promoter. Restriction sites were chosenthat were known to be absent in sequenced antibody genes.

Another use of the polyclonal and/or monoclonal antibodies of theinvention includes the stimulation of apoptosis by targeting thereceptor to Vip3A with antibodies. The interaction of antibodies raisedagainst cell surface-located proteins that are involved in controllingthe cell growth result in the induction of apoptosis by means ofpreventing the said receptor from binding to its natural ligand(s). Forinstance, the anti-APO-1 antibody completely blocks proliferation ofleukemia cells bearing the APO-1 protein and triggers apoptosis in thesecells (Trauth, B. C. et al. Science 245:301-305 (1989)). Also, theactivity resulting from the interaction between a given receptor and aligand is mimicked by substituting the ligand for antibodies raisedagainst the receptor. For instance, the addition of certain anti-Fasantibodies to cells bearing the Fas receptor in their cell surfaces willmediate apoptosis in a similar fashion as when the ligand of the Fasreceptor is added (Itoh, N. et al. Cell 66:233-243 (1991)).

The receptor to Vip3A(a) isolated from black cutworm shares homologywith a family of extracelular glycoproteins known as Tenascins, and inparticular with Tenascin-X (Bristow, J. et al. J. Cell Biol. 122:265-278(1993)). Tenascin-Xs are known to be involved in cell-to-cell adhesionand signaling. Lack of functionality of Tenascin-X either by mutation orby removal of the gene leads to lethality. Therefore, antibodies raisedagainst different domains of the receptor to Vip3A(a) either effectivelyblock the receptor from binding to its ligand(s) or mimic theinteraction of the Vip3A(a) protein triggering apoptosis. This approachis extended to different receptors with similar biological functions. Inthis sense, antibodies raised against insect cell receptors involved incrucial cell growth and interaction processes lead to induction ofapoptosis and are used as an strategy to control insects.

Screening for Novel Insecticidal Activities whose Mode of Action IsApoptosis

The materials described in this invention are used to screen forchemical ligands that have pesticidal properties triggering apoptoticresponses. Chemical ligands include small organic molecules, peptides,and proteins. In one embodiment of the invention, insect cell lines areused as model organisms for insects to screen for compounds that areinsecticidal as a consequence of their ability to induce apoptosis.These cell lines are handled in a high-throughput screening format wherethe cells are grown in multi-well plates and are exposed to a variety ofcompounds. Yeast is also used as a model organism. Using proceduresdescribed herein or known in the art, determining whether a compound ispesticidal as a consequence of inducing apoptosis is accomplished.

One means by which to identify compounds that trigger apoptoticresponses through interaction with a known receptor is to resort toidentified receptors involved in the signal transduction pathwaytriggered in apoptotic insect cell lines. These receptors aretransformed into heterologous cell lines creating isogenic lines withone of them containing a gene for expression of a specific receptor andanother one which does not either possess, or express, such a gene.These cell lines are handled in a high-throughput screening formatwhereby the transformed cell lines expressing the receptor have adifferential response against compounds that trigger apoptosis throughtheir specific interaction with said receptor.

Also encompassed by the present invention is the characterization ofbiochemical and/or molecular markers that specifically identify aninsect cell line undergoing apoptosis. For example, it is possible toisolate specific cDNAs induced during an apoptotic process in specificinsect cell lines. Although the death core pathway seems to bephylogenetically conserved (Nagata, S. Cell 88:355-365 (1997)), thesignal transduction pathway from the receptor to the death core pathwayis subject to variation across organisms. Messenger RNAs differentiallyexpressed in insect cells undergoing apoptosis are identified by anumber of techniques readily available such as differential display(Bauer, D. et al. Nucleic Acid Res. 21:4272-4280 (1993)) or subtractivelibraries (Sommer, H. et al. EMBO J. 9:605-613 (1990)). Thedifferentially expressed cDNA-encoded proteins are used as markers forapoptosis in specific insect cell lines.

Transgenic Plants Comprising a DNA Sequence Encoding a Protein of theVIP3 Class

A host plant expressing at least one of the sequences of the inventionhas enhanced resistance to attack by plant pests and is thus betterequipped to withstand crop losses associated with such attack. By plantis meant any plant species which can be genetically transformed bymethods known in the art. Methods known in the art for planttransformation are discussed below. Host plants include, but are notlimited to, those species previously listed as target crops.

Plant Expression Cassettes

Methodologies for the construction of plant expression cassettes as wellas the introduction of foreign DNA into plants are described in the art.Such expression cassettes may include promoters, terminators, enhancers,leader sequences, introns and other regulatory sequences operably linkedto the pesticidal protein coding sequence. It is further recognized thatpromoters or terminators of the VIP3 genes can be used in expressioncassettes.

Toxin genes derived from microorganisms may also differ from plantgenes. Plant genes differ from genes found in microorganisms in thattheir transcribed RNA does not possess defined ribosome binding sitesequence adjacent to the initiating methionine. Consequently, microbialgenes can be enhanced by the inclusion of a eukaryotic consensustranslation initiator at the ATG (Kozak, Cell 44:283-292 (1986)).Clontech (1993/1994 catalog, page 210) has suggested the sequenceGTCGACCATGGTC (SEQ ID NO:- - -) as a consensus translation initiator forthe expression of the E. coli uidA gene in plants. Further, Joshi(Nucleic Acids Res. 15: 6643-6653 (1987)) has compared many plantsequences adjacent to the ATG and suggests the consensus TAAACAATGGCT(SEQ ID NO:- - -). In situations where difficulties are encountered inthe expression of microbial ORFs in plants, inclusion of one of thesesequences at the initiating ATG may improve translation. In such casesthe last three nucleotides of the consensus may not be appropriate forinclusion in the modified sequence due to their modification of thesecond AA residue. Preferred sequences adjacent to the initiatingmethionine may differ between different plant species. By surveying thesequence of maize genes present in the GenBank/EMBL database it can bediscerned which nucleotides adjacent to the ATG should be modified toenhance translation of the toxin gene introduced into maize.

In addition, it has been shown that removal of illegitimate splice sitescan enhance expression and stability of introduced genes. Genes clonedfrom non-plant sources and not optimized for expression in plants maycontain motifs which can be recognized in plants as 5′ or 3′ splicesites. Consequently, the transcription process can be prematurelyterminated, generating truncated or deleted mRNA. The toxin genes can beengineered to remove these illegitimate splice sites using multipletechniques. For example, several available methods can be utilized toidentify potential splice sites in a DNA sequence. First, potentialsplice sites may be identified by computer analysis of the DNA sequence.Consensus sequences which identify splice sites are known in the art.See, for example, Goodall, G. J. and Filipowicz, W., EMBO J. 10,2635-2644 (1991) and Brown, J. W. S., Nucleic Acids Research 14,9549-9559 (1986). Alternately, one can identify splice sites actuallyprocessed by a plant by comparing PCR analysis of cDNA derived from thegene with actual gene products. Shorter than expected products areindicative of splicing. Such smaller products are then cloned andsequenced and the exact location of the splice determined. It is alsorecognized that a combination of computer searching and PCR analysis canbe utilized.

The novel toxin genes of the present invention, either as their nativesequence or as optimized synthetic sequences as described above, can beoperably fused to a variety of promoters for expression in plantsincluding constitutive, inducible, temporally regulated, developmentallyregulated, chemically regulated, tissue-preferred and tissue-specificpromoters to prepare recombinant DNA molecules, i.e., chimeric genes.Preferred constitutive promoters include the CaMV 35S and 19S promoters(Fraley et al., U.S. Pat. No. 5,352,605, issued Oct. 4, 1994). Anadditionally preferred promoter is derived from any one of several ofthe actin genes, which are known to be expressed in most cell types. Thepromoter expression cassettes described by McElroy et al. (Mol. Gen.Genet. 231: 150-160 (1991)) can be easily modified for the expression ofthe novel toxin gene and are particularly suitable for use inmonocotyledonous hosts.

Yet another preferred constitutive promoter is derived from ubiquitin,which is another gene product known to accumulate in many cell types.The ubiquitin promoter has been cloned from several species for use intransgenic plants (e.g. sunflower—Binet et al. Plant Science 79: 87-94(1991), maize—Christensen et al. Plant Molec. Biol. 12: 619-632 (1989)).The maize ubiquitin promoter has been developed in transgenic monocotsystems and its sequence and vectors constructed for monocottransformation are disclosed in the patent publication EP 0 342 926. Theubiquitin promoter is suitable for the expression of the novel toxingene in transgenic plants, especially monocotyledons.

Other promoters useful for the expression of the novel toxin gene inplants, particularly maize, are, for example, tissue-specific ortissue-preferential promoters such as those disclosed in WO 93/07278;chemically inducible promoters disclosed in EP-A 0 332 104, hereinincorporated by reference in its entirety.

In addition to promoters, a variety of transcriptional terminators arealso available for use in chimeric gene construction using the noveltoxin gene of the present invention. Transcriptional terminators areresponsible for the termination of transcription beyond the transgeneand its correct polyadenylation. Appropriate transcriptional terminatorsand those which are known to function in plants include the CaMV 35Sterminator, the tml terminator, the nopaline synthase terminator, thepea rbcS E9 terminator and others known in the art. These can be used inboth monocotyledons and dicotyledons.

A number of non-translated leader sequences derived from viruses such asthose reported in, for example, WO 96/10083 are also known to enhanceexpression, and these are particularly effective in dicotyledonouscells. Specifically, leader sequences from Tobacco Mosaic Virus (TMV,the “Ω-sequence”), Maize Chlorotic Mottle Virus (MCMV), and AlfalfaMosaic Virus (AMV) have been shown to be effective in enhancingexpression (e.g. Gallie et al. Nucl. Acids Res. 15: 8693-8711 (1987);Skuzeski et al. Plant Molec. Biol. 15; 65-79 (1990))

Various intron sequences have been shown to enhance expression whenadded to the 5′ regulatory region, particularly in monocotyledonouscells. For example, the introns of the maize Adh1 gene have been foundto significantly enhance the expression of the wild-type gene under itscognate promoter when introduced into maize cells (Callis et al., GenesDevelop. 1:1183-1200 (1987)).

Optimizing vip3 Genes for Plant Expression

The pesticidal genes of the invention can be optimized for enhancedexpression in plants. See, for example, EPA 0359472; EPA 0385962; WO91/16432; and, Perlak et al., Proc. Natl. Acad. Sci. 88:3324-3328(1991). In this manner, the coding sequences can be synthesized whichare optimized for plant expression.

In one embodiment of the invention the vip3A(a) is made according to theprocedure disclosed in U.S. Ser. No. 07/951,715, herein incorporated byreference. In this procedure, maize preferred codons, i.e., the singlecodon which most frequently encodes that amino acid in maize, are used.The maize preferred codon for a particular amino acid may be derived,for example, from known gene sequences from maize. Maize codon usage for28 genes from maize plants is found in Murray et al., Nucleic AcidsResearch 17: 477-498 (1989), the disclosure of which is incorporatedherein by reference. Examples of synthetic sequences made with maizeoptimized codons are set forth in SEQ ID NO:7 (VIP3A(a)), in SEQ IDNO:19 (VIP3A(b)), and in SEQ ID NO:20 (VIP3A(c)).

In this manner, the nucleotide sequences can be optimized for expressionin any plant. It is recognized that all or any part of the gene sequencemay be optimized or synthetic. That is, synthetic or partially optimizedsequences may also be used.

Plant Transformation

The recombinant DNA molecules can be introduced into the plant cell in anumber of art-recognized ways. Those skilled in the art will appreciatethat the choice of method might depend on the type of plant, i.e.monocot or dicot, targeted for transformation. Suitable methods oftransforming plant cells are described, for example, in WO 96/10083 andWO 97/46105, respectively, including microinjection, electroporation,Agrobacterium-mediated transformation, direct gene transfer, andballistic particle acceleration using devices available from Agracetus,Inc., Madison, Wis. and Dupont, Inc., Wilmington, Del.

An preferred embodiment is the protoplast transformation method formaize as disclosed in European Patent Application EP 0 292 435, as wellas in U.S. Pat. No. 5,350,689, hereby incorporated by reference in itsentirety. One particularly preferred set of embodiments for theintroduction of the expression cassettes of the present invention intowheat by microprojectile bombardment can be found in U.S. Pat. No.5,610,042 herein incorporated by reference in its entirety.

Transformation of plants can be undertaken with a single DNA molecule ormultiple DNA molecules (i.e. co-transformation), and both thesetechniques are suitable for use with the expression cassettes of thepresent invention. Numerous transformation vectors are available forplant transformation, and the expression cassettes of this invention canbe used in conjunction with any such vectors. The selection of vectorwill depend upon the preferred transformation technique and the targetspecies for transformation.

Many vectors are available for transformation using Agrobacteriumtumefaciens. These typically carry at least one T-DNA border sequenceand include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)). Inone preferred embodiment, the novel toxin gene of the present inventionmay be inserted into either of the binary vectors pCIB200 and pCIB2001for use with Agrobacterium, the construction of which is disclosed, forexample, in WO 95/33818 (example 35) (see also EP 0 332 104, example19).

An additional vector useful for Agrobacterium-mediated transformation isthe binary vector pCIB10 contains a gene encoding kanamycin resistancefor selection in plants, T-DNA right and left border sequences andincorporates sequences from the wide host-range plasmid pRK252 allowingit to replicate in both E. coli and Agrobacterium. Its construction isdescribed by Rothstein et al. (Gene 53: 153-161 (1987)). Variousderivatives of pCIB10 have been constructed which incorporate the genefor hygromycin B phosphotransferase described by Gritz at al. (Gene 25:179-188 (1983)). These derivatives enable selection of transgenic plantcells on hygromycin only (pCIB743), or hygromycin and kanamycin(pCIB715, pCIB717).

Methods using either a form of direct gene transfer orAgrobacterium-mediated transfer usually, but not necessarily, areundertaken with a selectable marker which may provide resistance to anantibiotic (e.g., kanamycin, hygromycin or methotrexate) or a herbicide(e.g., phosphinothricin). The choice of selectable marker for planttransformation is not, however, critical to the invention unless theexpression of this resistance and its biochemical activity interfereswith the choice of protoxin to toxin conversion chosen for use increating conditional fertility.

For certain plant species, different antibiotic or herbicide selectionmarkers may be preferred. Selection markers used routinely intransformation include the nptll gene which confers resistance tokanamycin and related antibiotics (Messing & Vierra, Gene 19: 259-268(1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene whichconfers resistance to the herbicide phosphinothricin (White et al., NuclAcids Res 18:1062 (1990), Spencer et al., Theor Appl Genet79:625-631(1990)), the hph gene which confers resistance to theantibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4:2929-2931), the dhfr gene, which confers resistance to methotrexate(Bourouis et al., EMBO J. 2: 1099-1104 (1983)), the mannose phosphate isisomerase gene, which allows selection on mannose as a carbon source (EP530 1 29,WO 94/20627).

One such vector useful for direct gene transfer techniques incombination with selection by the herbicide Basta (or phosphinothricin)is pCIB3064. This vector is based on the plasmid pCIB246, whichcomprises the CaMV 35S promoter in operational fusion to the E. coli GUSgene and the CaMV 35S transcriptional terminator and is described in thePCT published application WO 93/07278, herein incorporated by reference.Another useful selectable marker is obtained by operably linking aubiquitin promoter, a synthetic PAT gene and a nos terminator. Onceexample of a vector comprising this marker is the plasmid pCIB9804.

An additional transformation vector is pSOG35 which utilizes the E. coligene dihydrofolate reductase (DHFR) as a selectable marker conferringresistance to methotrexate and the construction of which is described,for example, in WO 95/33818 (example 35)

Another transformation vector is the vector pGL2 (Shimamoto et al.Nature 338, 274-276 (1989)) which contains the Streptomyces hygromycinphosphotransferase gene (hpt) operably linked to the 35S promoter and35S terminator sequences.

Transgenic plants can also be identified through the use of a scorablemarker. Examples of scorable markers useful in the invention areβ-glucuronidase, green fluorescent protein, and the C1 and β-peruregulatory genes of the maize anthocyanin pathway. In addition,transgenic plants expressing a VIP3 protein can be identified byscreening them for insecticidal activity without the need for eitherscorable or selectable markers.

Transformation of maize with a DNA sequence encoding a protein of theVIP3 class, but preferably a VIP3A(c) protein according to any of theabove methods can be readily achieved by microprojectile bombardment ofeither immature zygotic embryos or serially-propagatable Type Iembryogenic callus.

For transformation using immature zygotic embryos, ears areself-pollinated and immature zygotic embryos are obtained approximately10 days later. Approximately eight hundred immature zygotic embryos aredivided among different target plates containing a medium capable ofinducing and supporting the formation of embryogenic callus. Theimmature zygotic embryos are transferred immediately to the same mediumbut containing 12% sucrose. After 5 hours, the immature zygotic embryosare bombarded with a plasmid or plasmids using the PDS-1000/He devicefrom BioRad. The plasmid or plasmids comprise a selectable marker, suchas a gene conferring resistance to phosphinothricin, or a scorablemarker, such as green fluorescent protein, and a gene encoding a proteinof the VIP3 class prepared for delivery to and expression in maizeaccording to the above description. The plasmid or plasmids areprecipitated onto 1 μm gold particles essentially according to thepublished procedure from BioRad. The particles are delivered using aburst pressure of 1550 psi of helium. Each target plate is shot twicewith the plasmid and gold particle preparation. Since in one embodimentof the invention the plasmid or plasmids comprise a chimeric gene codingfor resistance to phosphinothricin this substance could be used toselect transformed cells in vitro. If used, the selection agent isapplied at 10 mg/L on the day of gene delivery and increased to 40 mg/Lafter approximately one month. The embryogenic callus so obtained may beregenerated in the presence of the selection agent phosphinothricin ifthe selectable marker is used. Plants are obtained from the selectedembryogenic callus lines. The regenerated plants are assayed forresistance to a susceptible insect. All the plants that are resistant tothe insect also express the introduced chimeric gene encoding a proteinor proteins of the VIP3 class as evidenced by the detection of VIP3protein in the plant using an ELISA assay. Plants resistant to theinsect and expressing the VIP3 protein are transformed.

For transformation of maize using Type I embryogenic callus, the callusis obtained from immature zygotic embryos using standard culturetechniques. For gene delivery, approximately 300 mg of the Type I callusis prepared by either chopping with a scalpel blade or by subculturing3-5 days prior to gene delivery. Prior to gene delivery, the preparedcallus is placed onto semi-solid culture medium again containing 12%sucrose. After approximately 4 hours, the tissue is bombarded using thePDS-1000/He Biolistic device from BioRad. The plasmid or plasmidscomprise a selectable marker, such as a gene conferring resistance tophosphinothricin, or a scorable marker, such as green fluorescentprotein, and a gene encoding a protein of the VIP3 class prepared fordelivery to and expression in maize according to the above description.The plasmids are precipitated onto 1 μm gold particles using essentiallythe standard protocol from BioRad. Approximately 16 hours after genedelivery the callus is transferred to standard culture medium containing2% sucrose and, if the selectable marker is used, to 1 mg/Lphosphinothricin. The callus is subcultured on selection for 8 weeks,after which surviving and growing callus is transferred to standardregeneration medium for the production of plants. The regenerated plantsare assayed for resistance to a susceptible insect. All the plants thatare resistant to the insect also express the introduced chimeric geneencoding a protein of the VIP3 class as evidenced by the detection ofVIP3 protein in the plant using an ELISA assay. Plants resistant to theinsect and expressing a protein of the VIP3 class are transformed.

Supplemental Insect Control Principles

The pesticidal proteins of the invention can be used in combination withBt δ-endotoxins or other insecticidal proteins to increase insect targetrange. Furthermore, the use of the VIPs of the present invention, butpreferably of the VIP3A(c) protein, in combination with Bt δ-endotoxinsor other insecticidal principles of a distinct nature has particularutility for the prevention and/or management of insect resistance.

The various insecticidal crystal proteins from Bacillus thuringiensishave been classified based upon their spectrum of activity and sequencesimilarity. The classification put forth by Höfte and Whiteley,Microbiol. Rev. 53: 242-255 (1989) placed the then known insecticidalcrystal proteins into four major classes. Generally, the major classesare defined by the spectrum of activity, with the Cry1 proteins activeagainst Lepidoptera, Cry2 proteins active against both Lepidoptera andDiptera, Cry3 proteins active against Coleoptera, and Cry4 proteinsactive against Diptera.

Within each major class, the δ-endotoxins are grouped according tosequence similarity. The Cry1 proteins are typically produced as 130-140kDa protoxin proteins which are proteolytically cleaved to produceactive toxin proteins about 60-70 kDa. The active portion of theδ-endotoxin resides in the NH₂-terminal portion of the full-lengthmolecule. Höfte and Whiteley, supra, classified the then known Cry1proteins into six groups, 1Aa, 1Ab, 1Ac, 1B, 1C, and 1D. Since then,proteins classified as Cry1Ea, Cry1Fa, Cry9A, Cry9C and Cry9B have alsobeen characterized.

The spectrum of insecticidal activity of an individual δ-endotoxin fromBacillus thuringiensis tends to be quite narrow, with a givenδ-endotoxin being active against only a few insects. Specificity is theresult of the efficiency of the various steps involved in producing anactive toxin protein and its subsequent ability to interact with theepithelial cells in the insect digestive tract. In one preferredembodiment, expression of VIPs in a transgenic plant is accompanied bythe expression of one or more Bt δ-endotoxins. Particularly preferred Btδ-endotoxins are those disclosed in U.S. application Ser. No.07/951,715, herein incorporated by reference.

It is well known that many δ-endotoxin proteins from Bacillusthuringiensis are actually expressed as protoxins. These protoxins aresolubilized in the alkaline environment of the insect gut and areproteolytically converted by proteases into a toxic core fragment (Höfteand Whiteley, Microbiol. Rev. 53: 242-255 (1989)). For δ-endotoxinproteins of the Cryl class, the toxic core fragment is localized in theN-terminal half of the protoxin. It is within the scope of the presentinvention that genes encoding either the full-length protoxin form orthe truncated toxic core fragment of the novel toxin proteins can beused in plant transformation vectors to confer insecticidal propertiesupon the host plant.

Other insecticidal principles include protease inhibitors (both serineand cysteine types), lectins, α-amylase, peroxidase and cholesteroloxidase. Other VIP genes, such as vip1A(a) and vip2A(a) as disclosed inU.S. Ser. No. 08/463,483 and herein incorporated by reference, are alsouseful in the present invention.

This co-expression of more than one insecticidal principle in the sametransgenic plant can be achieved by genetically engineering a plant tocontain and express all the genes necessary. Alternatively, a plant,Parent 1, can be genetically engineered for the expression of VIPs. Asecond plant, Parent 2, can be genetically engineered for the expressionof a supplemental insect control principle. By crossing Parent 1 withParent 2, progeny plants are obtained which express all the genesintroduced into Parents 1 and 2.

Recombinant Microorganisms Comprising Genes and Proteins of the VIP3Class

It is recognized that the isolated genes of the present invention whichencode a protein of the VIP3 class, but preferably a VIP3A(c) protein,can be transferred into any microbial host and confer their insecticidalproperties upon that host. Alternate hosts for the novel genes of thepresent invention can be selected as suitable for cloning purposes, forpurposes of characterizing the form and function of the gene or encodedprotein, for use as a fermentation host to increase production of thetoxin protein, for purposes of delivering at least one of the toxinproteins more effectively to the target insect pest, or introduction ofthe novel toxin gene into insect pathogens such as baculovirus (anuclear polyhedrosis virus, e.g. Autographica californica) to improvetheir effectiveness.

It is envisioned that said alternate host would be applied to theenvironment or plants or animals for insect control. Microorganism hostsmay be selected which are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one ormore crops of interest. These microorganisms are selected so as to becapable of successfully competing in the particular environment with thewild-type microorganisms, provide for stable maintenance and expressionof the gene expressing the polypeptide pesticide, and, desirably,provide for improved protection of the pesticide from environmentaldegradation and inactivation.

Such microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., Bacillus,Caulobacter, Agmenellum, Pseudomonas, Erwinia, Serratia, Klebsiella,Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylius,Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter,Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g.,Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula,and Aureobasidium. Of particular interest are such phytosphere bacterialspecies as Bacillus spp., Pseudomonas syringae, Pseudomonas fluorescens,Serratia marcescens, Acetobacter xylinum, Agrobacteria, Rhodopseudomonasspheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenesentrophus, Clavibacter xyli and Azotobacter vinlandii; and phytosphereyeast species such as Rhodotorula rubra, R. glutinis, R. marina, R.aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii,Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomycesrosues, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans.Of particular interest are the pigmented microorganisms.

Suitable host cells, where the pesticide-containing cells will betreated to prolong the activity of the toxin in the cell when the thentreated cell is applied to the environment of the target pest(s), mayinclude either prokaryotes or eukaryotes, normally being limited tothose cells which do not produce substances toxic to higher organisms,such as mammals. However, organisms which produce substances toxic tohigher organisms could be used, where the toxin is unstable or the levelof application sufficiently low as to avoid any possibility of toxicityto a mammalian host. As hosts, of particular interest will be theprokaryotes and the lower eukaryotes, such as fungi. Illustrativeprokaryotes, both Gram-negative and-positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae.Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, whichincludes yeast, such a Saccharomyces and Schizosaccharromyces; andBasidiomycetes yeast, such as Rhodotorula, Aureobasidium,Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing the protein gene intothe host, availability of expression systems, efficiency of expression,stability of the protein in the host, and the presence of auxiliarygenetic capabilities. Characteristics of interest for use as a pesticidemicrocapsule include protective qualities for the pesticide, such asthick cell walls, pigmentation, and intracellular packaging or formationof inclusion bodies; leaf affinity; lack of mammalian toxicity;attractiveness to pests for ingestion; ease of killing and fixingwithout damage to the toxin; and the like. Other considerations includeease of formulation and handling, economics, storage stability, and thelike.

Host organisms of particular interest include yeast, such as Rhodotorulasp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.;phylloplane organisms such as Pseudomonas sp., Erwinia sp. andFlavobacterium sp.; or such other organisms as Escherichia,LactoBacillus sp., Bacillus sp., and the like. Specific organismsinclude Pseudomonas aeurginosa, Pseudomonas fluorescens, Saccharomycescerevisiae, Bacillus thuringiensis, Escherichia coli, Bacillus subtilis,and the like.

A number of ways are available for introducing a gene expressing thepesticidal protein into the microorganism host under conditions whichallow for stable maintenance and expression of the gene. For example,expression cassettes can be constructed which include the DNA constructsof interest operably linked with the transcriptional and translationalregulatory signals for expression of the DNA constructs, and a DNAsequence homologous with a sequence in the host organism, wherebyintegration will occur, and/or a replication system which is functionalin the host, whereby integration or stable maintenance will occur.

Transcriptional and translational regulatory signals include but are notlimited to promoter, transcriptional initiation start site, operators,activators, enhancers, other regulatory elements, ribosomal bindingsites, an initiation codon, termination signals, and the like. See, forexample, U.S. Pat. Nos. 5,039,523; 4,853,331; EPO 0480762A2; Sambrook etal. supra; Molecular Cloning, a Laboratory Manual, Maniatis et al. (eds)Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982); AdvancedBacterial Genetics, Davis et al. (ads.) Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1980); and the references cited therein.

The novel genes or recombinant forms thereof can be transformed intosuch alternate hosts using a variety of art recognized methods. One suchpreferred method is electroporation of microbial cells, as described,for example, by the method of Dower (U.S. Pat. No. 5,186,800). Anotherpreferred method is that of Schurter et al. (Mol. Gen. Genet.218:177-181 (1989)), which is also disclosed in U.S. Ser. No. 07/353,565which is incorporated herein in its entirety.

Genes encoding the VIP3 class of proteins can be introduced intomicroorganisms that multiply on plants (epiphytes) or in plants(endophytes) to deliver proteins of the VIP3 class to potential targetpests. Many bacterial species are capable of living in the vasculartissues of plants. Most of these endophytes and epiphytes appear to havelittle physiological impact on plant growth and productivity.

Root colonizing bacteria, for example, can be isolated from the plant ofinterest by methods known in the art. Specifically, a Bacillus cereusstrain which colonizes roots could be isolated from roots of a plant(for example see J. Handelsman, S. Raffel, E. Mester, L. Wunderlich andC. Grau, Appl. Environ. Microbiol. 56:713-718, (1990)). Vip3 genes canalso be introduced into a root colonizing Bacillus cereus by standardmethods known in the art. Specifically, a gene encoding a protein of theVIP3 class derived from strain AB88 can be introduced into a rootcolonizing Bacillus cereus by means of conjugation using standardmethods (J. Gonzalez, B. Brown and B. Carlton, Proc. Natl. Acad. Sci.79:6951-6955, (1982)).

Also, the novel genes of the invention can be introduced into the rootcolonizing Bacillus by means of electro-transformation. For example,vip3A(a) can be cloned into a shuttle vector, for example, pHT3101 (D.Lereclus et al., FEMS Microbiol. Letts., 60:211-218 (1989)). The shuttlevector pHT3101 containing the coding sequence can then be transformedinto the root colonizing Bacillus by means of electroporation (D.Lereclus et al. 1989, FEMS Microbiol. Letts. 60:211-218). It is alsopossible to use the cotton colonizing Bacillus megaterium.

Another example is afforded by the endophyte Clavibacter xyli, which isfrom a genus/species known contain phytopathogenic bacteria which causeplant stunting. This bacterium can grow to very high levels in thevascular system of plants. A δ-endotoxin was introduced into thisendophyte, which when inoculated into a plant, provided good control ofcorn borer. Other endophytes are also known.

Expression systems can be designed so that VIP3 proteins are secretedoutside the cytoplasm of gram negative bacteria, E. coli, for example.Advantages of having VIP3 proteins secreted are (1) it can increase thelevel of VIP3 protein expressed and (2) can aid in efficientpurification of VIP3 protein.

VIP3 proteins can be made to be secreted in E. coli, for example, byfusing an appropriate E. coli signal peptide to the amino-terminal endof the VIP3 signal peptide or replacing the VIP3 signal peptide with theE. coli signal peptide. Signal peptides recognized by E. coli can befound in proteins already known to be secreted in E. coli, for examplethe OmpA protein (J. Ghrayeb, H. Kimura, M. Takahara, Y. Masui and M.Inouye, EMBO J., 3:2437-2442 (1984)). OmpA is a major protein of the E.coli outer membrane and thus its signal peptide is thought to beefficient in the translocation process. Also, the OmpA signal peptidedoes not need to be modified before processing as may be the case forother signal peptides, for example lipoprotein signal peptide (G.Duffaud, P. March and M. Inouye, Methods in Enzymology, 153:492 (1987)).

Specifically, unique BamHI restriction sites can be introduced at theamino-terminal and carboxy-terminal ends of the VIP coding sequencesusing standard methods known in the art. These BamHI fragments can becloned, in frame, into the vector pIN-III-ompA1, A2 or A3 (J. Ghrayeb,H. Kimura, M. Takahara, H. Hsiung, Y. Masui and M. Inouye, EMBO J.,3:2437-2442 (1984)) thereby creating ompA:VIP fusion gene which issecreted into the periplasmic space. The other restriction sites in thepolylinker of pIN-III-ompA can be eliminated by standard methods knownin the art so that the VIP3 amino-terminal amino acid coding sequence isdirectly after the ompA signal peptide cleavage site. Thus, the secretedVIP3 sequence in E. coli would then be identical to the native VIP3sequence.

When the VIP3 native signal peptide is not needed for proper folding ofthe mature protein, such signal sequences can be removed and replacedwith the ompA signal sequence. Unique BamHI restriction sites can beintroduced at the amino-termini of the proprotein coding sequencesdirectly after the signal peptide coding sequences of VIP3 and at thecarboxy-termini of VIP3 coding sequence. These BamHI fragments can thenbe cloned into the pIN-III-ompA vectors as described above.

General methods for employing the strains of the invention in pesticidecontrol or in engineering other organisms as pesticidal agents are knownin the art. See, for example U.S. Pat. No. 5,039,523 and EP 0480762A2.

VIP3 can be fermented in a bacterial host and the resulting bacteriaprocessed and used as a microbial spray in the same manner that Bacillusthuringiensis strains have been used as insecticidal sprays. In the caseof a VIP3 which is secreted from Bacillus, the secretion signal isremoved or mutated using procedures known in the art. Such mutationsand/or deletions prevent secretion of the VIP3 protein(s) into thegrowth medium during the fermentation process. The VIP3 proteins areretained within the cell and the cells are then processed to yield theencapsulated VIP3 protein. Any suitable microorganism can be used forthis purpose. Psuedomonas has been used to express Bacillusthuringiensis endotoxins as encapsulated proteins and the resultingcells processed and sprayed as an insecticide. (H. Gaertner et al. 1993,In Advanced Engineered Pesticides, L. Kim ed.)

Various strains of Bacillus thuringiensis are used in this manner. SuchBt strains produce endotoxin protein(s) as well as VIP3. Alternatively,such strains can produce only VIP3. A sporulation deficient strain ofBacillus subtilis has been shown to produce high levels of the Cry3Aendotoxin from Bacillus thuringiensis (Agaisse, H. and Lereclus, D.,“Expression in Bacillus subtilis of the Bacillus thuringiensis CryIIIAtoxin gene is not dependent on a sporulation-specific sigma factor andis increased in a spoOA mutant”, J. Bacteriol., 176:4734-4741 (1994)). Asimilar spoOA mutant can be prepared in Bacillus thuringiensis and usedto produce encapsulated VIP3 which are not secreted into the medium butare retained within the cell.

Target crops to be protected within the scope of the present inventioncomprise, e.g., the following species of plants:

cereals (wheat, barley, rye, oats, rice, sorghum and related crops),beet (sugar beet and fodder beet), forage grasses (orchardgrass, fescue,and the like), drupes, pomes and soft fruit (apples, pears, plums,peaches, almonds, cherries, strawberries, raspberries and blackberries),leguminous plants (beans, lentils, peas, soybeans), oil plants (rape,mustard, poppy, olives, sunflowers, coconuts, castor oil plants, cocoabeans, groundnuts), cucumber plants (cucumber, marrows, melons) fiberplants (cotton, flax, hemp, jute), citrus fruit (oranges, lemons,grapefruit, mandarins), vegetables (spinach, lettuce, asparagus,cabbages and other Brassicae, onions, tomatoes, potatoes, paprika),lauraceae (avocados, carrots, cinnamon, camphor), deciduous trees andconifers (e.g. linden-trees, yew-trees, oak-trees, alders, poplars,birch-trees, firs, larches, pines), or plants such as maize, tobacco,nuts, coffee, sugar cane, tea, vines, hops, bananas and natural rubberplants, as well as ornamentals (including composites).

The microorganisms which have been genetically altered to contain thepesticidal gene and protein may be used for protecting agriculturalcrops and products from pests. In one aspect of the invention, whole,i.e., unlysed, cells of a toxin (pesticide)-producing organism aretreated with reagents that prolong the activity of the toxin produced inthe cell when the cell is applied to the environment of target pest(s).

Alternatively, the pesticides are produced by introducing a heterologousgene into a cellular host. Expression of the heterologous gene results,directly or indirectly, in the intracellular production and maintenanceof the pesticide. These cells are then treated under conditions thatprolong the activity of the toxin produced in the cell when the cell isapplied to the environment of target pest(s). The resulting productretains the toxicity of the toxin. These naturally encapsulatedpesticides may then be formulated in accordance with conventionaltechniques for application to the environment hosting a target pest,e.g., soil, water, and foliage of plants. See, for example EPA 0192319,and the references cited therein.

The active ingredients of the present invention are normally applied inthe form of compositions and can be applied to the crop area or plant tobe treated, simultaneously or in succession, with other compounds. Thesecompounds can be both fertilizers or micronutrient donors or otherpreparations that influence plant growth. They can also be selectiveherbicides, insecticides, fungicides, bactericides, nematicides,mollusicides or mixtures of several of these preparations, if desired,together with further agriculturally acceptable carriers, surfactants orapplication-promoting adjuvants customarily employed in the art offormulation. Suitable carriers and adjuvants can be solid or liquid andcorrespond to the substances ordinarily employed in formulationtechnology, e.g. natural or regenerated mineral substances, solvents,dispersants, wetting agents, tackifiers, binders or fertilizers.

Preferred methods of applying an active ingredient of the presentinvention or an agrochemical composition of the present invention whichcontains at least one of the pesticidal proteins produced by thebacterial strains of the present invention are leaf application, seedcoating and soil application. The number of applications and the rate ofapplication depend on the intensity of infestation by the correspondingpest.

Entomocidal Compositions Comprising a Recombinant Bacillus thuringiensisStrain

The present invention further provides an entomocidal compositioncomprising a recombinant Bacillus thuringiensis strain containing atleast one of the novel toxin genes in recombinant form, or derivativesor mutants thereof, together with an agricultural adjuvant such as acarrier, diluent, surfactant or application-promoting adjuvant. Thecomposition may also contain a further biologically active compoundselected from fertilizers, micronutrient donors, plant growthpreparations, herbicides, insecticides, fungicides, bactericides,nematicides and molluscicides and mixtures thereof. The composition maycomprise from 0.1 to 99% by weight of a recombinant Bacillusthuringiensis strain containing at least one of the novel genes inrecombinant form, or the derivatives or mutants thereof, from 1 to 99.9%by weight of a solid or liquid adjuvant, and from 0 to 25% by weight ofa surfactant. The recombinant Bacillus thuringiensis strain containingat least one of the novel genes in recombinant form, or the compositioncontaining it, may be administered to the plants or crops to beprotected together with certain other insecticides or chemicals (1993Crop Protection Chemicals Reference, Chemical and Pharmaceutical Press,Canada) without loss of potency. It is compatible with most othercommonly used agricultural spray materials but should not be used inextremely alkaline spray solutions. It may be administered as a dust, asuspension, a wettable powder or in any other material form suitable foragricultural application.

A recombinant Bacillus thuringiensis strain containing at least one ofthe novel genes in recombinant form is normally applied in the form ofentomocidal compositions and can be applied to the crop area or plant tobe treated, simultaneously or in succession, with further biologicallyactive compounds. These compounds may be both fertilizers ormicronutrient donors or other preparations that influence plant growth.They may also be selective herbicides, insecticides, fungicides,bactericides, nematicides, molluscicides or mixtures of several of thesepreparations, it desired together with further carriers, surfactants orapplication-promoting adjuvants customarily employed in the art offormulation. Suitable carriers and adjuvants can be solid or liquid andcorrespond to the substances ordinarily employed in formulationtechnology, e.g. natural or regenerated mineral substances, solvents,dispersants, wetting agents, tackifiers, binders or fertilizers. Theformulations, i.e. the entomocidal compositions, preparations ormixtures containing the recombinant Bacillus thuringiensis straincontaining the novel gene in recombinant form as an active ingredient orcombinations thereof with other active ingredients, and, whereappropriate, a solid or liquid adjuvant, are prepared in known manner,e.g., by homogeneously mixing and/or grinding the active ingredientswith extenders, e.g., solvents, solid carriers, and in some casessurface-active compounds (surfactants).

Solvents, carriers, surfactants, surface active compounds, etc that arecustomarily employed in the art of formulation and can be suitably usedwithin the present invention are disclosed, for example, in WO 96/10083.

Another particularly preferred characteristic of an entomocidalcomposition of the present invention is the persistence of the activeingredient when applied to plants and soil. Possible causes for loss ofactivity include inactivation by ultra-violet light, heat, leaf exudatesand pH. For example, at high pH, particularly in the presence ofreductant, δ-endotoxin crystals are solubilized and thus become moreaccessible to proteolytic inactivation. High leaf pH might also beimportant, particularly where the leaf surface can be in the range of pH8-10. Formulation of an entomocidal composition of the present inventioncan address these problems by either including additives to help preventloss of the active ingredient or encapsulating the material in such away that the active ingredient is protected from inactivation.Encapsulation can be accomplished chemically (McGuire and Shasha, 1992)or biologically (Barnes and Cummings, 1986). Chemical encapsulationinvolves a process in which the active ingredient is coated with apolymer while biological encapsulation involves the expression of theδ-endotoxin genes in a microbe. For biological encapsulation, the intactmicrobe containing the δ-endotoxin protein is used as the activeingredient in the formulation. The addition of UV protectants mighteffectively reduce irradiation damage. Inactivation due to heat couldalso be controlled by including an appropriate additive.

The entomocidal compositions usually contain 0.1 to 99%, preferably 0.1to 95%, of a recombinant Bacillus thuringiensis strain containing atleast one of the novel genes in recombinant form, or combination thereofwith other active ingredients, 1 to 99.9% of a solid or liquid adjuvant,and 0 to 25%, preferably 0.1 to 20%, of a surfactant. Whereas commercialproducts are preferably formulated as concentrates, the end user willnormally employ dilute formulations of substantially lowerconcentration. The entomocidal compositions may also contain furtheringredients, such as stabilizers, antifoams, viscosity regulators,binders, tackifiers as well as fertilizers or other active ingredientsin order to obtain special effects.

Methods of Controlling Insects

In view of the above description of the invention, it is apparent thatthere are several methods by which insects may be controlled usingproteins of the VIP3 class as an insecticidal principle, either alone orin combination with supplementary insect control principles such asδ-endotoxins. Any method of delivering a VIP3 protein for ingestion by asusceptible insect will result in the control of that insect.

In one embodiment of the invention, plants are transformed with a geneencoding a protein of the VIP3 class. Expression of the protein mayoccur at any time during growth and development of the plant, dependingon the nature of the insect to be controlled. For example, a protein ofthe VIP3 class can, according to the invention, be expressed in roots,stems, leaves, seeds, pollen, etc. This provides the advantage ofexpressing the protein only in those cells or tissues upon which thetarget insect feeds. Feeding the cells or tissues of a plant expressingVIP3 protein to a susceptible insect will result in the control of thatinsect. In one embodiment of the invention, a VIP3 protein is expressedin the stem or stalk of a plant in order to control black cutworm. Theplants may be grown under either field or greenhouse conditions. Seedcontaining a VIP3 protein can also be protected against insect damagewhen in storage.

EXAMPLES

Examples 16 to 18 on pages 73 to 82 of WO 96/10083 describe theisolation and biological characterization of Bacillus thuringiensisstrains AB88 and AB424, the purificatin and characterization of aVIP3A(a) protein, the cloning of vip3A(a) and vip3A(b) genes, and theidentification of new vip genes by hybridization. Said Examples areincorporated herein in their entirety by reference. The followingexamples further describe the materials and methods used in carrying outthe invention and the subsequent results. They are offered by way ofillustration, and their recitation should not be considered as alimitation of the claimed invention.

Example 1

Presence of vip3-like genes and VIP3-like proteins in Bacillus isolates

Bacillus isolates other than AB88 have demonstrated insecticidalactivity against Lepidopteran larvae when spent culture supernatantswere tested. Some isolates which were active against black cutworm wereanalyzed for the presence of vip3-like genes and for the production ofVIP3-like proteins.

A standard PCR analysis was used to determine whether the blackcutworm-active Bacillus isolates contained a vip3-like gene. Using thePCR primer pair GW110 (5′-CGA TTA ATG TTG GCC TC-3′; SEQ ID NO:17) andGW111 (5′-CAT TAG CAT CTC CGG ACA CAG-3′; SEQ ID NO:18) it wasdetermined that all of the black cutworm active isolates produced a 728bp vip3 gene product which was equal to the size produced by the typestrain, AB88. One Bacillus isolate, AB51, which was not active againstblack cutworm, produced the same size vip3 product. None of the othernon-black cutworm active Bacillus isolates produced a vip3 PCR product.

Analysis of VIP3 protein production was done using a standard westernblot procedure. Antibodies raised against the VIP3A(a) protein describedin the above example were used to detect immunoreactive proteins.Aliquots of cell free culture supernatants from sporulated cultures wererun on SDS-PAGE gels using standard methods. Standard western blottingprocedures were then carried out to determine the presence of VIP3-likeproteins. All of the Bacillus isolates which had a 728 bp PCR productand were active against black cutworm produced an 80 kDa protein whichwas immunoreactive to the VIP3A(a) antibody. The AB51 isolate which hadthe correct size vip3 PCR product but was not active against blackcutworm produced an immunoreactive protein which was truncatedsuggesting this may be the reason no biological activity against blackcutworm was observed.

Example 2

Characterization of Bacillus thuringiensis Strain AB51 Containing avip3-like Gene

A B. thuringiensis strain, designated AB51, was shown to containproteins of the VIP3 class by western analysis using rabbit polyclonalanti-Vip3A(a) antibodies. The vip3-like gene was cloned into pKS whichcreated pCIB7112. This gene was given the designation vip3A(c). The DNAsequence for vip3A(c) is disclosed in SEQ ID NO:5 and the encodedprotein sequence is disclosed in SEQ ID NO:6. The VIP3A(c) protein is746 amino acids long, 43 amino acids shorter than its VIP3A(a) andVIP3A(b) homologues.

Example 3

Development of Antibodies to VIP3A(a) Protein

Antiserum against purified Vip3A(a) insecticidal protein was produced inrabbits and goats. For rabbits, nitrocellulose-bound protein (50 μg) wasdissolved in DMSO, emulsified with Freund's complete adjuvant (Difco)and injected subcutaneously twice a month for three months. For goats,active soluble pure Vip3A protein (300 μg) was injected intramuscularlytwice a month for three month. They were bled 10 days after the secondand third injection and the serum was recovered from the blood sample(Harlow, E. and Lane, D. Antibodies: A Manual Laboratory, Cold SpringHarbor Lab. Press, NY, 1988). The antiserums were then fractionated byaffinity chromatography utilizing staphylococcal protein A, and theresulting IgG fraction was further purified by filtering through acolumn containing immobilized-E. coli lysate (Yu, C. G. et al. Appl.Environ. Microbiol. 63:532-536 (1997)).

The rabbit and goat antiserums were characterized analyzing the Vip3A(a)protein by western blot. Proteins were separated by SDS/PAGE andtransferred to nitrocellulose. Nitrocellulose blots were blocked in 20mM Tris-HCl, pH 7.5/0.15 M NaCl/0.02% NaN_(3/5)% nonfat dry milk. Blotswere developed by using either rabbit raised or goat-raisedanti-Vip3A(a) antibodies at a concentration of 200 ng/ml or 100 ng/mlrespectively. Alkaline phosphatase-conjugated goat antirabbit IgG orrabbit antigoat antiserum were used as secondary antibodies at aconcentration of 1 μg/ml (Kirkegaard & Perry Laboratories, Inc.).Bromochloroindolyl-phosphate and nitroblue tetrazolium were used assubstrate for the alkaline phosphatase reaction. Both anti-Vip3A(a)antibodies, the rabbit and the goat raised, are polyclonal. Theanti-Vip3A(a) antibodies obtained from goat have a higher titer than theones obtained from rabbits. In the experimental approach, anti-Vip3A(a)antibodies from rabbit should be used at a dilution {fraction (1/500)}from the original serum (200 ng/ml). By comparison, the anti-Vip3A(a)antibodies obtained from goat can be diluted up to {fraction (1/2000)}(100 ng/ml) from the original serum. While the rabbit raised antibodiesonly recognize the N-terminal portion of the Vip3A(a) protein, theantibodies obtained from goats react with epitopes present throughoutthe full length of the Vip3A(a) protein.

Example 4

Construction of Plant Expression Cassettes

Plant expression cassettes consist of promoters that can drive theexpression of a coding sequence either constitutively or in atissue-specific manner, the coding sequences to be expressed and thetermination sequences which allow the polyadenylation of the mRNA andits proper translation.

The promoters selected in the DNA constructs of the present inventionincludes constitutive promoters such as the one from the maize ubiquitingene (Christensen et al. Plant Mol. Biol. 12:619-632, 1989) (pCIB8029,FIG. 4; pCIB8055, FIG. 5; pCIB9806, FIG. 6), and tissue specificpromoters such as those from the maize methalothionein-like gene (deFramond, A. FEBS 290:103-106, 1991) (pCIB8030, FIG. 7; pCIB8056,pCIB9805) which provides a root-preferred expression, from the maizePEPC gene (Hudspeth, R. L. and Grula, J. W. Plant Mol. Biol.12:579-589,1989) (pCIB5535, FIG. 8; pCIB9807) which provides agreen-tissue specific expression, and from the barley non-specific lipidtransfer protein LTP4 (pCIB9819, FIG. 9)(Molina, A. and Garcia-Olmedo,F. Plant J. 4:983-991, 1993) which provides a stem-preferred expression.All constructs used in the present invention contain the terminatorsequence derived from the 35S CaMV and the intron 9 derived from themaize PEPC gene for enhancing gene expression purposes. The plasmidspCIB8029, pCIB8055, and pCIB9806 contain the intron#1 of the maizeubiquitin gene placed between the maize ubiquitin promoter and thevip3A(a) gene. The construct comprising the encoding sequence of thevip3A(a) gene, the intron#9 and the 35S terminator sequence wasengineered into the recipient plasmid bearing the different promoters asdouble digests BamHI-EcoRI.

The plant expression cassettes were used as such in the planttransformation experiments, or they were linearised by using restrictionenzymes that cut in the Amp^(R) gene of the backbone plasmid. In someexperiments, fragments comprising the promoter, gene of interest, intronand terminator were isolated from the rest of the plasmid backbone byrestriction digestion and fragment purification. In these cases fragmentpurification proceeded as follows: 500 ug of DNA is digested with theappropriate enzyme and separated on a 0.8% agarose gel. The fragment ofinterest is identified, cut out from the gel and purified using aDurapore Millipore filter (0.45 micron). The filtrate containing thefragment is precipitated with sodium acetate and ethanol. The fragmentis resuspended in TE and used in transformation experiments.

Example 5

Insecticidal Activity of Maize Plants Expressing VIP3A(a)

Maize plants expressing VIP3A(a) protein were tested for insecticidaleffects on the insect species listed in the table below by the followingprocedure. One to four 4 cm sections were cut from leaves of transgenicand control maize plants. Each leaf piece was placed on a moistenedfilter disc in a 50×9 mm petri dish. Five neonates of the species beingtested were placed on each leaf piece giving a total of 5-20 larvaetested for each plant. The Petri dishes were incubated at 30° C. in thedark. Mortality was scored after 48-72 hours. Results are shown in Table11.

TABLE 11 Percent mortality Insect species tested VIP3A(a) Control MaizePests Black cutworm (Agrotis ipsilon) 100 0 Fall armyworm (Spodopterafrugiperda) 100 0 Sugarcane borer (Diatrea saccharalis) 100 0Southwestern corn borer (Diatrea grandiosella) 100 0 Corn earworm(Helicoverpa zea) 100 10 Mediterranean corn borer (Sesamia nonagroides)100 15 Other Lepidopteran Pests Beet armyworm (S. exigua) 100 0 Yellowstriped armyworm (S. ornithogalli) 100 0 Cabbage looper (Trichoplusiani) 100 20

Example 6

Expression of vip3A(a) in Maize Plants

Transformation of maize elite Ciba inbred lines CG00526 and 2154 withthe Vip3 gene was achieved using particle bombardment of Type I callustissue. For transformation using Type I embryogenic callus, the calluswas obtained from zygotic embryos using standard culture techniques andsubcultured 1-2 days prior to bombardment. Callus tissue was preparedfor bombardment by placing ˜20, 3-5 mm diameter pieces arranged in aring shape onto culture medium containing 12% sucrose. Callus tissue wasplaced onto this media for four hours prior to bombardment. DNA used fortransformation of maize callus was either circular plasmid DNA, linearplasmid DNA, or purified DNA fragments containing the Vip3 gene undercontrol of various plant promoters. In experiments where a selectableagent was used, the gene allowed resistance to phosphinothricin orallowed for growth in the presence of mannose. Plasmids or DNA fragmentsisolated by filtration were precipitated onto 0.3 um gold particlesaccording to published procedures from BioRad Laboratories, Hercules,Calif. Gold particles were delivered using a burst pressure of 650 psiof helium. Each target plate was shot twice with the DNA coatedparticles. Sixteen to twenty hours after bombardment the CG00526 calluswas transferred to standard culture maintenance media. Seven dayspost-bombardment the tissue was transferred to media containing theselection agent, Basta at a concentration of 100 mg/L. Basta is acommercial formulation of glufosinate ammonium produced by Hoechst.Callus of 2154 was kept on 12% sucrose for 1-7 days after bombardmentand transferred to standard culture media containing 20-30 mg/L Basta atday 7. The 2154 and CG00526 callus was subcultured in the presence of 30or 100 mg/L Basta, respectively, for eight weeks. Tissue survivingselection was subcultured onto lower levels of Basta (5-40 mg/L) for aperiod of approximately five to ten weeks to allow for tissue bulk-upand then transferred to a standard regeneration media with no selectionfor the production of plants. Commonly, 12% of the callus piecesbombarded produced transformed callus that survived Basta selection.Individual transformed calli would typically be regenerated to produce20-30 plants.

Events were generated from experiments where no selection was used. Inthese experiments the callus was grown for a period of 9-10 weeks onmaintenance media prior to transferring to regeneration media. Event1337 is an example of a transformed VIP3 event derived from atransformation experiment with no selectable or scorable marker byscreening plants for insecticidal activity.

Transformed calli were also generated from experiments where mannoseselection was used. In these transformations the phosphomannoseisomerase gene under control of the maize ubiquitin promoter of pCIB9818was bombarded with the Vip3 gene. Mannose at 0.5-1.5% was included inthe maintenance media for a period of twelve weeks and not included inthe regeneration media.

Transgenic plants were evaluated for VIP3A(a) protein expression byinsect bioassay and ELISA assay. Leaf pieces were removed from 2-4 leafstage plants for evaluation using both black cutworm and fall army wormbioassays. Bioassays were done using ten newly hatched larvae placed indishes with leaf pieces. Percent mortality was calculated at 72 hours.Tissues from transgenic plants were also assayed by ELISA using standardprotocols to quantitate Vip3 protein levels in different plant tissues.Plant tissue was extracted and Table 17 provides representative eventsgenerated and their corresponding of insect bioassay results.

Transgenic maize plants were transformed with various plasmidscontaining the Vip3 gene under control of various promoters such as themaize PEP-carboxylase promoter (PEPC), the maize ubiquitin promoter(Ubi), and the maize metallothionein-like promoter (MTL). The selectablemarker gene was the PAT gene under control of the maize ubiquitinpromoter in pUBIAC. Representative events listed in Table 12 show theevents produced with different plasmids or DNA fragments derived fromplasmids. DNA fragments were generated using restriction enyzmedigestions and size fractionated using electrophoresis in 0.8% agarosegels. The DNA fragments were excised from the gels, frozen, crushed andpurified by filtration through 0.45 micron DuraPore Millipore filtersfollowed by ethanol precipitation. Transformed maize events weregenerated with circular plasmid DNA of pCIB5535 containing the Vip3 geneunder control of the maize PEPC promoter. Events were also transformedwith linear plasmid DNA of pCIB5535 and pCIB8029 containing the Vip3gene under control of the maize ubiquitin promoter. Additional eventswere produced by bombarding purified DNA restriction enzyme fragmentscontaining just the Vip3 gene with promoter. Fragments corresponding tothe Vip3 gene include: a 4906 bp EcoRI/HindIII fragment from pCIB5535with the maize PEPC promoter, a 5142 bp KpnI/HindIII fragment frompCIB8030 with the MTL promoter; a 4597 bp KpnI/HindIII fragment ofpCIB8029 with the maize ubiquitin promoter; a 4818 bp HindIII fragmentof pCIB8055 with the maize ubiqutin promoter; a 5364 HindIII fragment ofpCIB8056 with the MTL promoter; a 5964 Ascl fragment of pCIB9805 withthe MTL promoter; a 5418 bp Ascl fragment of pCIB9806 with the maizeubiqutin promoter; and a 5727 bp Ascl fragment of pCIB9807 with themaize PEPC promoter.

TABLE 12 Mortality (%) Fall Black Event No. Plasmid Used Chimeric GeneArmyworm Cutworm 891 pCIB5535 PEPC:vip3A(a) 100 100 906 pCIB5535 andPEPC:vip3A(a) 100 100 pCIB8029 and Ubi:vip3A(a) 946 pCIB5535 andPEPC:vip3A(a) 100 100 pCIB8030 and MTL:vip3A(a)

Example 7

Insecticidal Activity of Maize Plants Containing Vip3 and Btδ-endotoxins

VIP3A(a) has little activity against European corn borer (ECB). To makeplants with broad spectrum lepidopteran control, maize plants containinga vip3A(a) gene were crossed with maize plants containing a cry1B, whichis active against ECB. Progeny from the crosses were bioassayed againstECB and fall armyworm (FAW) as described in Example 1. Results are shownin Table 13. Approximately 34% of the progeny were not active againsteither species, 15.4% were active only on ECB, 23.1% were active only onFAW and 27.9% were active against both species. Plants active againstboth species contained both VIP3A(a) and Cry1B protein. Similar resultsare obtained using other Bt δ-endotoxins, particularly Cry1Ab or Cry9C.

TABLE 13 % ECB % FAW % ECB & FAW Cross active active active % not activeVIP3A(a) X Cry1B 15.4 23.1 27.9 34.6

Example 8

VIP3A(a) Lyses the Midgut Epithelial Cells of Susceptible Insects

Feeding and gut clearance studies. The temporal sequence of symptomsfollowing the ingestion of VIP3A(a)-containing diet by second-instarblack cutworm (BCW) larvae, a susceptible insect, were recorded from thetime of initial administration until larval death. Larvae exposed tocontrol diet showed active feeding followed by uninterrupted gutparastalsis. In contrast, the addition of VIP3A(a) protein in the diethad a significant effect on feeding behavior. When added atconcentrations as low as 4 ng per cm², the larvae fed on and off duringperiods of 10-20 min. The presence of blue color in their guts indicatedfeeding but the clearance of the gut content was dramatically affectedas judged by the deceased number of frass. With 4 ng of VIP3A(a) per cm²added to the diet, larval development was significantly impaired after a48 h incubation period but no mortality was observed. At concentrationsof 40 ng of Vip3A(a) per cm², the larvae suffered gut paralysis uponingestion of minute amounts of diet and no frass could be seenindicating an almost complete lack of gut clearance. Under thiscondition, ca. 50% mortality was recorded after 48 hr. Whenconcentrations higher than 40 ng of VIP3A(a) per cm² were used, thelarvae were moribund after only a few bites, with no frass and mortalityrates approaching 100%. When similar experiments were conducted withfall armyworm, also a susceptible insect, similar behavioral patternswere observed. In contrast, European corn borer did not alter itsfeeding behavior when VIP3A(a) protein was added to the diet even atconcentrations as high as 400 ng of VIP3A(a) per cm².

Histological observations of the effects of the Vip3A(a) protein.Histopathological observations on the effects of the VIP3A(a) protein onBCW were conducted on second and third instar larvae which had been feda diet containing VIP3A(a). Analysis of BCW gut cross-sections showedextensive damage to the midgut epithelium indicating that the midguttissue is a primary site of action of the Vip3A(a) protein. No damagewas discernible in the foregut and hindgut. Midgut epithelial cells fromuntreated larvae were closely associated with one another showing noevidence of damage. Sections from larvae that had been fed for 24 h withdiet containing Vip3A(a) showed that distal ends of the epitheliumcolumnar cells had become distended and bulbous. Although the gobletcells exhibited some morphological alterations, they did not show signsof damage at this stage. Degeneration of the epithelium columnar cellscontinued such that, after 48 h of ingesting Vip3A(a)-containing diet,the lumen was filled with debris of disrupted cells. The goblet cellsalso exhibited signs of damage after 48 h, but both types of cells werestill attached to the basement membrane. Black cutworm larvae were deadat 72 h and desquamation of the epithelial layer was complete. While asimilar histopathology was observed for fall armyworm, European cornborer did not exhibit any tissue damage under similar experimentalconditions.

In vivo immunolocalization of the Vip3A(a) protein. Third instar larvaeof black cutworm and European corn borer fed on artificial dietsupplemented with 100-200 ng of VIP3A(a) per cm² were used forimmunocytochemical characterization of the VIP3A(a) binding to midgutsections. The bound VIP3A(a) was visualized using rabbit anti-VIP3A(a)antibodies previously purified through protein A sepharose and E. coliimmobilized columns (Yu, C. G. et al. Appl. Environ. Microbiol.63:532-536, 1997). VIP3A(a) binding was detected in midgut epithelium ofblack cutworm, while showing no binding to European corn borer midguts.Midgut sections from black cutworm larvae fed with control diet showedno VIP3A(a) binding. The VIP3A(a) binding seems to be specificallyassociated to the apical microvilli and it is mostly associated to thecolumnar cells, with no detectable signal in the goblet cells.

Example 9

VIP3A(a) and ViP3A(b) Induce Apoptosis in Insect Cells

VIP3A(a) and VIP3A(b) were shown to be a apoptosis inducing proteinarose by the characterization of its insecticidal effects towards aninsect cell line (Sf-9) derived from Spodoptera frugiperda, an insectsusceptible to VIP3A(a). VIP3A(a) showed insecticidal activity towardsthe insect cell line when kept present throughout the experiment. WhenSF-9 insect cells are transiently exposed to VIP3A(a) and VIP3A(b),their cell viability was significantly reduced even with exposure timesas short as 5 min. Once the incubation time exceeded 10 min, the effectsof the VIP3A(a) and VIP3A(b) on insect cell viability over a period of 6hours were maximal showing a reduction of 90% in cell viability. Thecytological changes occurring in SF-9 cells transiently exposed toVIP3A(a) were monitored by microscopy. Small protrusions appeared on thesurface of the treated cells some time between 10 and 15 min after theirexposure to the VIP3A(a) protein. At this stage, the mitochondria of thecells remained functionally intact as revealed by staining withrhodamine 123, a dye that accumulates in mitochondria with activemembrane potential (Johnson, L. V. et al. Proc. Natl. Acad. Sci. USA77:990-994, 1980). These protrusions eventually disappeared and thecells entered a phase of profuse vacuolization lasting an additional 30to 60 min. During the final stages, the insect cells are seen to swellbefore disintegration. For an individual cell, the entire processrequired 1 to 2 hours. All these cellular events are consistent withprevious studies on cells undergoing apoptosis particularly consideringthat programmed cell death during metamorphosis of certain insects isaccompanied by cellular vacuolization and swelling (Schwartz, L. M. etal. Proc. Natl. Acad. Sci. USA 90:980-984 (1993)).

Recent studies have shown that the distribution of phospholipids in theplasma membrane is affected in very early stages of animal cellsundergoing apoptosis (Martin, S. J., et al. J. Exp. Med.:182, 1545-1556,1995) particularly the externalization of the phosphatidylserine (PS).This process can be visualized by using Annexin V, an anticoagulantprotein with high affinity for phosphatidylserine (PS). WhenVIP3A(a)-treated SF-9 cells were incubated with Annexin V, anexternalization of PS was revealed in insect cell membranes as early as5-10 min after the exposure to VIP3A(a) probably marking the onset ofapoptosis.

One of the key molecular events that is the hallmark of apoptosis isendonucleolysis resulting in a double strand DNA break freeingoligonucleosome-sized fragments of 200 base pair and multiples. Weexamined the occurrence of endonucleolysis in SF-9 cells treated withVIP3A(a) using an in situ detection method and analysing the DNA byagarose gel electrophoresis. Based on the ability of the Klenow enzymeto incorporated modified nucleotides using the DNA ends generated by DNAfragmentation, SF-9 insect cells showed signs of endonucleolysis asearly as 30 min upon their exposure to the VIP3A(a) protein. This stagewill coincide with the appearance of membrane-bound subcellularapoptotic bodies visualized in the microscopical observations. Theseearly indications of endonucleolytic activity were confirmed by thedetection of DNA fragments in agarose gels characteristic of a chromatinladder slightly latter in the process. These results corroborated theindications obtained from cytological observations, that the SF-9 cellsinitiate an apoptotic-type of programmed cell death upon their exposureto the VIP3A(a) protein.

The VIP3A(a) and VIP3A(b) proteins were discovered on the basis of theirinsecticidal properties against some lepidopteran insects. Therefore, wewere interested in knowing whether the VIP3A(a) protein would induce anapoptotic pathway in gut cells of susceptible insects upon its ingestionand thus, it could exert its insecticidal properties by triggering anactive process of cell death. Histological and histochemical studieshave shown that the VIP3A(a) protein specifically targets the columnarcells of the midgut epithelium of susceptible insects provoking cellchanges characterized by membrane protrusions and extensivevacuolization leading to cell death. These cytological changes inducedby VIP3A(a) in insect gut cells resemble those described above for SF-9cells. We then examined whether midgut epithelium cells of susceptibleinsects undergo endonucleolysis upon ingesting diet containing VIP3A(a)by in situ detection (Cuvillier, O., et al. Nature 381:800-803 (1996))of DNA fragmentation. When sections of midgut tissue from black cutwormlarvae fed with diet either containing VIP3A(a) or control diet, nucleistaining indicative of DNA fragmentation was only detectable in thecolumnar cells of the midgut epithelium exposed to the VIP3A(a) protein.This result indicates that the VIP3A(a) protein induces anendonucleolysis process in the midgut epithelium cells concurrently withthe cytological changes reported previously. It is our conclusion thatthe VIP3A(a) protein likely exerts its insecticidal properties byactivating an apoptosis-type of programmed cell death of the midgutepithelium cells of susceptible insects.

Example 10

Isolation of the Receptor for VIP3A(a) from Black Cutworm

Black cutworm is sensitive to VIP3A(a) and therefore this insect wasused for the isolation of the VIP3A(a) receptor. Midgut of third instarblack cutworm larvae were collected by dissection and immediately frozenin liquid nitrogen. One gram of midgut tissue was used to isolate mRNAby following the protocol described in the two-hybrid cDNA libraryconstruction kit provided by Clontech (1997). Ten micrograms of poly A⁺RNA were used as starting material. In first strand synthesis, bothrandom and lock-docking oligo(dT)₂₅d(A/C/G) primers are used in separatesynthesis with MML reverse transcriptase. The second strand cDNA wasachieved by an optimal ratio of DNA polymerase to Rnase H activity inthe second-strand enzyme cocktail. The newly synthesized double strandedcDNA is then ligated to EcoRI-NotI-SalI adaptors. The cDNAs were ligatedinto pGAD10 (Vijaychander, S. et al. CLONTECHniques IX-3:8-10, 1994)which provides the activation domain. The vip3A(a) gene was engineeredinto the polylinker site of the plasmid pGBT9 in frame with the GAL4-DNAbinding domain (Bartel, P. L. et al. Cellular Interactions inDevelopment: A Practical Approach, pp.153-179, Oxford University Press,1993). The recombinant pGBT9-vip3A(a) was transformed into the yeaststrain GGY1::171 (Gill, G. and Ptashne, M. Cell51:121-126, 1987) byelectroporation (Estruch, J. J. et al. BioTechniques 16:610-612, 1994).The transformed yeast was selected in minimal media withouttryptophan(Bartel, P. L. et al. Cellular Interactions in Development: APractical Approach, pp. 153-179, Oxford University Press, 1993). Theexpression of the VIP3A(a) protein in the recombinant yeast wasconfirmed by western analysis. The yeast strain GGY1::171- VIP3A(a) wastransformed with the black cutworm cDNA library represented in pGAD10.GGY1::171 possess the HIS3 marker under the control of GAL4 recognitionsites. The HIS3 gene allows a positive growth selection for clones thatare transformed by two interacting hybrid constructs. After plating morethan 200,000 recombinant clones, only one was able to grow in minimalmedia without histidine. The plasmid DNA of the positive yeast colonywas isolated by the yeast lysis buffer method (Kaiser, P. and Auer, B.BioTechniques 14:552 (1993)) and electroporated into E. coli. The insertcontaining the cDNA was subcloned into the EcoRI site of the pBluescript(Stratagene) and sequenced by the dideoxy termination method of Sangeret al., Proc. Natl. Acad. Sci. USA, 74: 5463-5467 (1977), using PRISMReady Reaction Dye Deoxy Terminator Cycle Sequencing Kits and PRISMSequenase® Terminator Double-Stranded DNA Sequencing Kit and analysed onan ABI 373 automatic sequencer.

An alternative approach to identify clones encoding for protein(s) thatinteract with Vip3A consisted of plating yeast transformed with theblack cutworm cDNA library represented in pGAD10. After transferringsaid transformed yeast population to nitrocellulose filters, they werescreened with a biotin-labelled Vip3A protein followed by an incubationwith a solution containing streptavidin coupled to alkaline phosphatase(AP). After extensive washes, the clone or clones expressing a proteinwith the ability to bind to Vip3A were visualized by using the substrate5-bromo-4-chloro-3-indolyl phosphate (BCIP) in combination with nitroblue tetrazolium (NBT). AP catalizes the formation of insolubleprecipitates which developed in dark spots on the nitrocellulosefilters. The experimental procedures are described in detail bySambrook, J. et al. in Molecular Cloning: A Laboratory Manual, pp.12.21-12.24, Cold Spring Harbor Laboratory, 1989. After re-growing theyeast plates overnight, the developed spots in the filters were matchedto yeast colonies in the original plates. These colonies were grown,their plasmids isolated, and their inserts characterized as describedabove.

Example 11

Insect Cells Transformed with the Gene for the Receptor ExhibitApoptosis when Exposed to the VIP3A(a) Protein

The receptor in black cutworm midgut cells for the VIP3A(a) protein wascloned into the XhoI-BamHI site of the Smart 2 cosmid vector (Speek, M.et al Gene 64: 173-177 (1988)), and the recombinant construct was usedto transform the Schneider 2 (S2) Drosophila cell line using the calciumphosphate co-precipitation method (Clem, R. J. and Miller. L. K. Mol.Cel. Biol.14: 5212-5222 (1994)). Smart 2 carries the selectable markertet (tetracycline) for bacterial transformation and the neo (neomycin)for Drosophila cell transformation. The neo selectable marker isexpressed under the control of the Drosophila hsp70 promoter. Thetransformed S2 cells were selected in S2 Drosophila medium supplementedwith 10% of Fetal Seroalbumin and with G418 (1 mg/ml) at 30° C. (seeGIBCO catalogue 1997). Several stably transformed S2 cell lines wereestablished after 45 days of selection in the medium described above.

The sensitivity of the S2 transformed cells to the VIP3A(a) was testedby adding VIP3A(a) protein (at a final concentration of 1,7 microgramsper ml) to the media containing the transformed S2 cells that have beenpreviously heat shocked at 42° C. 30 min. The induction of apoptosis intransformed S2 cells was confirmed by both microscopical observationsand by the TACS Kit, and in situ Apoptosis Detection kit (for detaileddescription, see Trevigen catalogue 1996).

Example 12

Isolating Homologues to the Receptor from Other Insects

The cells of the midgut epithelium of black cutworm larvae possess areceptor that is recognized by the VIP3A(a) protein. Receptors fromother insects known to be susceptible to VIP3A(a) are isolated byidentifying the DNA sequences in Southern analysis. DNA is prepared,enzyme restricted, run in agarose gels and blotted onto nitrocelluloseand/or nylon filters. These filters are probed with the cDNA encodingthe receptor from black cutworm using low stringency conditions ofhybridization and washing. Genes with a similarity to the black cutwormreceptor to VIP3A(a) lower than 50% were identified. The Southernanalysis can also be probed against partial sequences of the cDNA whichencode specific domains such as death domain or EGF-like motifs with theintention of isolating genes that contain similar domains even thoughthey are functionally different to the black cutworm receptor toVIP3A(a).

The isolation of homologues to the black cutworm receptor to VIP3A(a) isbe accomplished by the two hybrid system described in Fields, S. andSong, O.-K. Nature 340:245-246 (1989). Isolated mRNA is obtained from anorganisms of interest, synthesize cDNAs and clone them into pGAD10 orequivalent plasmids. The cDNA library is co-transformed with thepGB9-bearing the vip3A(a) gene (or homologues of this gene) and rescuedputative receptors in yeast by means of activating a marker based uponprotein-protein interaction between the VIP3A(a) protein (or homologues)and the putative receptor.

Homologues to the black cutworm receptor to VIP3A(a) are isolated byexpressing cDNAs libraries isolated from organisms of interests, clonedinto appropriate expression vectors and transformed into host cells suchas yeast or insect cells which are known not to have the ability to bindand/or be sensitive to VIP3A(a). The transformed cells are screenedbased on their gained property of binding VIP3A(a) or undergoingapoptotic responses when incubated with VIP3A(a). In this case, theprotein VIP3A(a) is used as probe and its binding will be monitoredeither by antibodies against VIP3A(a) or by labels such as biotinattached to VIP3A(a).

Example 13

Screening for Novel Compounds that Induce Apoptosis in Insect Cells

Model cell lines for different orders of insects (some examples includeSf-9 cells for lepidopteran, Colorado potato beetle for coleopterans, S2from Drosophila for dipterans) is used to screen for novel compoundswhose mode of action is induction of apoptosis. The cells are grown inmulti-well plates which are used for a high-throughput assay screeningfor thousands of compounds (both of large and small molecular weight).The compound(s) are added as single component or as mixtures.Compound(s) inducing apoptosis are identified as follows: 1) membraneprotrusions are visible in the cell membrane, 2) a reorganization of thephosphatidylserine containing membrane lipids is detectable by usingspecific proteins with high affinity for phosphatidylserine such asAnnexin-V linked to a visual marker, 3) cytoplasmic blebbing is visiblein the cell cytoplasm, 4) active mitochondria can be visualized by usingvital dyes such as rhodamine 123 that accumulate in mitochondria, 5) DNAfragmentation is detected either by DNA analysis in agarose gels, byELISA detection of nucleosomal release or by in vivo detection of DNAnicking. All these cytological and molecular features are indicative ofapoptosis.

The black cutworm receptor to VIP3A(a) is transformed into S2 cell line.Therefore, isogenic S2 lines are available with and without the saidreceptor. These cell lines are used to screen compounds that provide adifferential response due to the presence of the said receptor.Transformed S2 cells undergoing apoptosis upon exposure to certaincompounds are identified as indicated above. The differential responseof the transformed versus the non-transformed cell is indicative thatthe action of the compound is mediated by the cloned receptor. Similarapproaches are undertaken with insect cells transformed with receptorshomologue to the black cutworm receptor to VIP3A(a).

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

22 1 2378 DNA Bacillus thuringiensis CDS (9)..(2375) Native DNA sequenceencoding VIP3A(a) protein from AB88 as contained in pCIB7104 1 agatgaacatg aac aag aat aat act aaa tta agc aca aga gcc tta cca 50 Met Asn LysAsn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro 1 5 10 agt ttt att gat tatttt aat ggc att tat gga ttt gcc act ggt atc 98 Ser Phe Ile Asp Tyr PheAsn Gly Ile Tyr Gly Phe Ala Thr Gly Ile 15 20 25 30 aaa gac att atg aacatg att ttt aaa acg gat aca ggt ggt gat cta 146 Lys Asp Ile Met Asn MetIle Phe Lys Thr Asp Thr Gly Gly Asp Leu 35 40 45 acc cta gac gaa att ttaaag aat cag cag tta cta aat gat att tct 194 Thr Leu Asp Glu Ile Leu LysAsn Gln Gln Leu Leu Asn Asp Ile Ser 50 55 60 ggt aaa ttg gat ggg gtg aatgga agc tta aat gat ctt atc gca cag 242 Gly Lys Leu Asp Gly Val Asn GlySer Leu Asn Asp Leu Ile Ala Gln 65 70 75 gga aac tta aat aca gaa tta tctaag gaa ata tta aaa att gca aat 290 Gly Asn Leu Asn Thr Glu Leu Ser LysGlu Ile Leu Lys Ile Ala Asn 80 85 90 gaa caa aat caa gtt tta aat gat gttaat aac aaa ctc gat gcg ata 338 Glu Gln Asn Gln Val Leu Asn Asp Val AsnAsn Lys Leu Asp Ala Ile 95 100 105 110 aat acg atg ctt cgg gta tat ctacct aaa att acc tct atg ttg agt 386 Asn Thr Met Leu Arg Val Tyr Leu ProLys Ile Thr Ser Met Leu Ser 115 120 125 gat gta atg aaa caa aat tat gcgcta agt ctg caa ata gaa tac tta 434 Asp Val Met Lys Gln Asn Tyr Ala LeuSer Leu Gln Ile Glu Tyr Leu 130 135 140 agt aaa caa ttg caa gag att tctgat aag ttg gat att att aat gta 482 Ser Lys Gln Leu Gln Glu Ile Ser AspLys Leu Asp Ile Ile Asn Val 145 150 155 aat gta ctt att aac tct aca cttact gaa att aca cct gcg tat caa 530 Asn Val Leu Ile Asn Ser Thr Leu ThrGlu Ile Thr Pro Ala Tyr Gln 160 165 170 agg att aaa tat gtg aac gaa aaattt gag gaa tta act ttt gct aca 578 Arg Ile Lys Tyr Val Asn Glu Lys PheGlu Glu Leu Thr Phe Ala Thr 175 180 185 190 gaa act agt tca aaa gta aaaaag gat ggc tct cct gca gat att ctt 626 Glu Thr Ser Ser Lys Val Lys LysAsp Gly Ser Pro Ala Asp Ile Leu 195 200 205 gat gag tta act gag tta actgaa cta gcg aaa agt gta aca aaa aat 674 Asp Glu Leu Thr Glu Leu Thr GluLeu Ala Lys Ser Val Thr Lys Asn 210 215 220 gat gtg gat ggt ttt gaa ttttac ctt aat aca ttc cac gat gta atg 722 Asp Val Asp Gly Phe Glu Phe TyrLeu Asn Thr Phe His Asp Val Met 225 230 235 gta gga aat aat tta ttc gggcgt tca gct tta aaa act gca tcg gaa 770 Val Gly Asn Asn Leu Phe Gly ArgSer Ala Leu Lys Thr Ala Ser Glu 240 245 250 tta att act aaa gaa aat gtgaaa aca agt ggc agt gag gtc gga aat 818 Leu Ile Thr Lys Glu Asn Val LysThr Ser Gly Ser Glu Val Gly Asn 255 260 265 270 gtt tat aac ttc tta attgta tta aca gct ctg caa gcc caa gct ttt 866 Val Tyr Asn Phe Leu Ile ValLeu Thr Ala Leu Gln Ala Gln Ala Phe 275 280 285 ctt act tta aca aca tgccga aaa tta tta ggc tta gca gat att gat 914 Leu Thr Leu Thr Thr Cys ArgLys Leu Leu Gly Leu Ala Asp Ile Asp 290 295 300 tat act tct att atg aatgaa cat tta aat aag gaa aaa gag gaa ttt 962 Tyr Thr Ser Ile Met Asn GluHis Leu Asn Lys Glu Lys Glu Glu Phe 305 310 315 aga gta aac atc ctc cctaca ctt tct aat act ttt tct aat cct aat 1010 Arg Val Asn Ile Leu Pro ThrLeu Ser Asn Thr Phe Ser Asn Pro Asn 320 325 330 tat gca aaa gtt aaa ggaagt gat gaa gat gca aag atg att gtg gaa 1058 Tyr Ala Lys Val Lys Gly SerAsp Glu Asp Ala Lys Met Ile Val Glu 335 340 345 350 gct aaa cca gga catgca ttg att ggg ttt gaa att agt aat gat tca 1106 Ala Lys Pro Gly His AlaLeu Ile Gly Phe Glu Ile Ser Asn Asp Ser 355 360 365 att aca gta tta aaagta tat gag gct aag cta aaa caa aat tat caa 1154 Ile Thr Val Leu Lys ValTyr Glu Ala Lys Leu Lys Gln Asn Tyr Gln 370 375 380 gtc gat aag gat tcctta tcg gaa gtt att tat ggt gat atg gat aaa 1202 Val Asp Lys Asp Ser LeuSer Glu Val Ile Tyr Gly Asp Met Asp Lys 385 390 395 tta ttg tgc cca gatcaa tct gaa caa atc tat tat aca aat aac ata 1250 Leu Leu Cys Pro Asp GlnSer Glu Gln Ile Tyr Tyr Thr Asn Asn Ile 400 405 410 gta ttt cca aat gaatat gta att act aaa att gat ttc act aaa aaa 1298 Val Phe Pro Asn Glu TyrVal Ile Thr Lys Ile Asp Phe Thr Lys Lys 415 420 425 430 atg aaa act ttaaga tat gag gta aca gcg aat ttt tat gat tct tct 1346 Met Lys Thr Leu ArgTyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser 435 440 445 aca gga gaa attgac tta aat aag aaa aaa gta gaa tca agt gaa gcg 1394 Thr Gly Glu Ile AspLeu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala 450 455 460 gag tat aga acgtta agt gct aat gat gat ggg gtg tat atg ccg tta 1442 Glu Tyr Arg Thr LeuSer Ala Asn Asp Asp Gly Val Tyr Met Pro Leu 465 470 475 ggt gtc atc agtgaa aca ttt ttg act ccg att aat ggg ttt ggc ctc 1490 Gly Val Ile Ser GluThr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu 480 485 490 caa gct gat gaaaat tca aga tta att act tta aca tgt aaa tca tat 1538 Gln Ala Asp Glu AsnSer Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr 495 500 505 510 tta aga gaacta ctg cta gca aca gac tta agc aat aaa gaa act aaa 1586 Leu Arg Glu LeuLeu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys 515 520 525 ttg atc gtcccg cca agt ggt ttt att agc aat att gta gag aac ggg 1634 Leu Ile Val ProPro Ser Gly Phe Ile Ser Asn Ile Val Glu Asn Gly 530 535 540 tcc ata gaagag gac aat tta gag ccg tgg aaa gca aat aat aag aat 1682 Ser Ile Glu GluAsp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn 545 550 555 gcg tat gtagat cat aca ggc gga gtg aat gga act aaa gct tta tat 1730 Ala Tyr Val AspHis Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr 560 565 570 gtt cat aaggac gga gga att tca caa ttt att gga gat aag tta aaa 1778 Val His Lys AspGly Gly Ile Ser Gln Phe Ile Gly Asp Lys Leu Lys 575 580 585 590 ccg aaaact gag tat gta atc caa tat act gtt aaa gga aaa cct tct 1826 Pro Lys ThrGlu Tyr Val Ile Gln Tyr Thr Val Lys Gly Lys Pro Ser 595 600 605 att cattta aaa gat gaa aat act gga tat att cat tat gaa gat aca 1874 Ile His LeuLys Asp Glu Asn Thr Gly Tyr Ile His Tyr Glu Asp Thr 610 615 620 aat aataat tta gaa gat tat caa act att aat aaa cgt ttt act aca 1922 Asn Asn AsnLeu Glu Asp Tyr Gln Thr Ile Asn Lys Arg Phe Thr Thr 625 630 635 gga actgat tta aag gga gtg tat tta att tta aaa agt caa aat gga 1970 Gly Thr AspLeu Lys Gly Val Tyr Leu Ile Leu Lys Ser Gln Asn Gly 640 645 650 gat gaagct tgg gga gat aac ttt att att ttg gaa att agt cct tct 2018 Asp Glu AlaTrp Gly Asp Asn Phe Ile Ile Leu Glu Ile Ser Pro Ser 655 660 665 670 gaaaag tta tta agt cca gaa tta att aat aca aat aat tgg acg agt 2066 Glu LysLeu Leu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser 675 680 685 acggga tca act aat att agc ggt aat aca ctc act ctt tat cag gga 2114 Thr GlySer Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gln Gly 690 695 700 ggacga ggg att cta aaa caa aac ctt caa tta gat agt ttt tca act 2162 Gly ArgGly Ile Leu Lys Gln Asn Leu Gln Leu Asp Ser Phe Ser Thr 705 710 715 tataga gtg tat ttt tct gtg tcc gga gat gct aat gta agg att aga 2210 Tyr ArgVal Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg Ile Arg 720 725 730 aattct agg gaa gtg tta ttt gaa aaa aga tat atg agc ggt gct aaa 2258 Asn SerArg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala Lys 735 740 745 750gat gtt tct gaa atg ttc act aca aaa ttt gag aaa gat aac ttt tat 2306 AspVal Ser Glu Met Phe Thr Thr Lys Phe Glu Lys Asp Asn Phe Tyr 755 760 765ata gag ctt tct caa ggg aat aat tta tat ggt ggt cct att gta cat 2354 IleGlu Leu Ser Gln Gly Asn Asn Leu Tyr Gly Gly Pro Ile Val His 770 775 780ttt tac gat gtc tct att aag taa 2378 Phe Tyr Asp Val Ser Ile Lys 785 2789 PRT Bacillus thuringiensis 2 Met Asn Lys Asn Asn Thr Lys Leu Ser ThrArg Ala Leu Pro Ser Phe 1 5 10 15 Ile Asp Tyr Phe Asn Gly Ile Tyr GlyPhe Ala Thr Gly Ile Lys Asp 20 25 30 Ile Met Asn Met Ile Phe Lys Thr AspThr Gly Gly Asp Leu Thr Leu 35 40 45 Asp Glu Ile Leu Lys Asn Gln Gln LeuLeu Asn Asp Ile Ser Gly Lys 50 55 60 Leu Asp Gly Val Asn Gly Ser Leu AsnAsp Leu Ile Ala Gln Gly Asn 65 70 75 80 Leu Asn Thr Glu Leu Ser Lys GluIle Leu Lys Ile Ala Asn Glu Gln 85 90 95 Asn Gln Val Leu Asn Asp Val AsnAsn Lys Leu Asp Ala Ile Asn Thr 100 105 110 Met Leu Arg Val Tyr Leu ProLys Ile Thr Ser Met Leu Ser Asp Val 115 120 125 Met Lys Gln Asn Tyr AlaLeu Ser Leu Gln Ile Glu Tyr Leu Ser Lys 130 135 140 Gln Leu Gln Glu IleSer Asp Lys Leu Asp Ile Ile Asn Val Asn Val 145 150 155 160 Leu Ile AsnSer Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Ile 165 170 175 Lys TyrVal Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr 180 185 190 SerSer Lys Val Lys Lys Asp Gly Ser Pro Ala Asp Ile Leu Asp Glu 195 200 205Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp Val 210 215220 Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gly 225230 235 240 Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu LeuIle 245 250 255 Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly AsnVal Tyr 260 265 270 Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Ala Gln AlaPhe Leu Thr 275 280 285 Leu Thr Thr Cys Arg Lys Leu Leu Gly Leu Ala AspIle Asp Tyr Thr 290 295 300 Ser Ile Met Asn Glu His Leu Asn Lys Glu LysGlu Glu Phe Arg Val 305 310 315 320 Asn Ile Leu Pro Thr Leu Ser Asn ThrPhe Ser Asn Pro Asn Tyr Ala 325 330 335 Lys Val Lys Gly Ser Asp Glu AspAla Lys Met Ile Val Glu Ala Lys 340 345 350 Pro Gly His Ala Leu Ile GlyPhe Glu Ile Ser Asn Asp Ser Ile Thr 355 360 365 Val Leu Lys Val Tyr GluAla Lys Leu Lys Gln Asn Tyr Gln Val Asp 370 375 380 Lys Asp Ser Leu SerGlu Val Ile Tyr Gly Asp Met Asp Lys Leu Leu 385 390 395 400 Cys Pro AspGln Ser Glu Gln Ile Tyr Tyr Thr Asn Asn Ile Val Phe 405 410 415 Pro AsnGlu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Lys 420 425 430 ThrLeu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly 435 440 445Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Tyr 450 455460 Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Val 465470 475 480 Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu GlnAla 485 490 495 Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser TyrLeu Arg 500 505 510 Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu ThrLys Leu Ile 515 520 525 Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Val GluAsn Gly Ser Ile 530 535 540 Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala AsnAsn Lys Asn Ala Tyr 545 550 555 560 Val Asp His Thr Gly Gly Val Asn GlyThr Lys Ala Leu Tyr Val His 565 570 575 Lys Asp Gly Gly Ile Ser Gln PheIle Gly Asp Lys Leu Lys Pro Lys 580 585 590 Thr Glu Tyr Val Ile Gln TyrThr Val Lys Gly Lys Pro Ser Ile His 595 600 605 Leu Lys Asp Glu Asn ThrGly Tyr Ile His Tyr Glu Asp Thr Asn Asn 610 615 620 Asn Leu Glu Asp TyrGln Thr Ile Asn Lys Arg Phe Thr Thr Gly Thr 625 630 635 640 Asp Leu LysGly Val Tyr Leu Ile Leu Lys Ser Gln Asn Gly Asp Glu 645 650 655 Ala TrpGly Asp Asn Phe Ile Ile Leu Glu Ile Ser Pro Ser Glu Lys 660 665 670 LeuLeu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gly 675 680 685Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gln Gly Gly Arg 690 695700 Gly Ile Leu Lys Gln Asn Leu Gln Leu Asp Ser Phe Ser Thr Tyr Arg 705710 715 720 Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg Ile Arg AsnSer 725 730 735 Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala LysAsp Val 740 745 750 Ser Glu Met Phe Thr Thr Lys Phe Glu Lys Asp Asn PheTyr Ile Glu 755 760 765 Leu Ser Gln Gly Asn Asn Leu Tyr Gly Gly Pro IleVal His Phe Tyr 770 775 780 Asp Val Ser Ile Lys 785 3 2612 DNA Bacillusthuringiensis CDS (118)..(2484) Native DNA sequence encoding VIP3A(b)from AB424 3 attgaaattg ataaaaagtt atgagtgttt aataatcagt aattaccaataaagaattaa 60 gaatacaagt ttacaagaaa taagtgttac aaaaaatagc tgaaaaggaagatgaac 117 atg aac aag aat aat act aaa tta agc aca aga gcc tta cca agtttt 165 Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe1 5 10 15 att gat tat ttc aat ggc att tat gga ttt gcc act ggt atc aaagac 213 Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp20 25 30 att atg aac atg att ttt aaa acg gat aca ggt ggt gat cta acc cta261 Ile Met Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asp Leu Thr Leu 3540 45 gac gaa att tta aag aat cag cag cta cta aat gat att tct ggt aaa309 Asp Glu Ile Leu Lys Asn Gln Gln Leu Leu Asn Asp Ile Ser Gly Lys 5055 60 ttg gat ggg gtg aat gga agc tta aat gat ctt atc gca cag gga aac357 Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu Ile Ala Gln Gly Asn 6570 75 80 tta aat aca gaa tta tct aag gaa ata tta aaa att gca aat gaa caa405 Leu Asn Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ala Asn Glu Gln 8590 95 aat caa gtt tta aat gat gtt aat aac aaa ctc gat gcg ata aat acg453 Asn Gln Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile Asn Thr 100105 110 atg ctt cgg gta tat cta cct aaa att acc tct atg ttg agt gat gta501 Met Leu Arg Val Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Val 115120 125 atg aaa caa aat tat gcg cta agt ctg caa ata gaa tac tta agt aaa549 Met Lys Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu Ser Lys 130135 140 caa ttg caa gag att tct gat aag ttg gat att att aat gta aat gta597 Gln Leu Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val Asn Val 145150 155 160 ctt att aac tct aca ctt act gaa att aca cct gcg tat caa aggatt 645 Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Ile165 170 175 aaa tat gtg aac gaa aaa ttt gag gaa tta act ttt gct aca gaaact 693 Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr180 185 190 agt tca aaa gta aaa aag gat ggc tct cct gca gat att cgt gatgag 741 Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Ala Asp Ile Arg Asp Glu195 200 205 tta act gag tta act gaa cta gcg aaa agt gta aca aaa aat gatgtg 789 Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp Val210 215 220 gat ggt ttt gaa ttt tac ctt aat aca ttc cac gat gta atg gtagga 837 Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gly225 230 235 240 aat aat tta ttc ggg cgt tca gct tta aaa act gca tcg gaatta att 885 Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu LeuIle 245 250 255 act aaa gaa aat gtg aaa aca agt ggc agt gag gtc gga aatgtt tat 933 Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn ValTyr 260 265 270 aac ttc cta att gta tta aca gct ctg caa gca aaa gct tttctt act 981 Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Ala Lys Ala Phe LeuThr 275 280 285 tta aca cca tgc cga aaa tta tta ggc tta gca gat att gattat act 1029 Leu Thr Pro Cys Arg Lys Leu Leu Gly Leu Ala Asp Ile Asp TyrThr 290 295 300 tct att atg aat gaa cat tta aat aag gaa aaa gag gaa tttaga gta 1077 Ser Ile Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe ArgVal 305 310 315 320 aac atc ctc cct aca ctt tct aat act ttt tct aat cctaat tat gca 1125 Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro AsnTyr Ala 325 330 335 aaa gtt aaa gga agt gat gaa gat gca aag atg att gtggaa gct aaa 1173 Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val GluAla Lys 340 345 350 cca gga cat gca ttg att ggg ttt gaa att agt aat gattca att aca 1221 Pro Gly His Ala Leu Ile Gly Phe Glu Ile Ser Asn Asp SerIle Thr 355 360 365 gta tta aaa gta tat gag gct aag cta aaa caa aat tatcaa gtc gat 1269 Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gln Asn Tyr GlnVal Asp 370 375 380 aag gat tcc tta tcg gaa gtt att tat ggc gat atg gataaa tta ttg 1317 Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp LysLeu Leu 385 390 395 400 tgc cca gat caa tct gga caa atc tat tat aca aataac ata gta ttt 1365 Cys Pro Asp Gln Ser Gly Gln Ile Tyr Tyr Thr Asn AsnIle Val Phe 405 410 415 cca aat gaa tat gta att act aaa att gat ttc actaaa aaa atg aaa 1413 Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr LysLys Met Lys 420 425 430 act tta aga tat gag gta aca gcg aat ttt tat gattct tct aca gga 1461 Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp SerSer Thr Gly 435 440 445 gaa att gac tta aat aag aaa aaa gta gaa tca agtgaa gcg gag tat 1509 Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser GluAla Glu Tyr 450 455 460 aga acg tta agt gct aat gat gat ggg gtg tat atgccg tta ggt gtc 1557 Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met ProLeu Gly Val 465 470 475 480 atc agt gaa aca ttt ttg act ccg att aat gggttt ggc ctc caa gct 1605 Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly PheGly Leu Gln Ala 485 490 495 gat gaa aat tca aga tta att act tta aca tgtaaa tca tat tta aga 1653 Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys LysSer Tyr Leu Arg 500 505 510 gaa cta ctg cta gca aca gac tta agc aat aaagaa act aaa ttg atc 1701 Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys GluThr Lys Leu Ile 515 520 525 gtc ccg cca agt ggt ttt att agc aat att gtagag aac ggg tcc ata 1749 Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Val GluAsn Gly Ser Ile 530 535 540 gaa gag gac aat tta gag ccg tgg aaa gca aataat aag aat gcg tat 1797 Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala Asn AsnLys Asn Ala Tyr 545 550 555 560 gta gat cat aca ggc gga gtg aat gga actaaa gct tta tat gtt cat 1845 Val Asp His Thr Gly Gly Val Asn Gly Thr LysAla Leu Tyr Val His 565 570 575 aag gac gga gga att tca caa ttt att ggagat aag tta aaa ccg aaa 1893 Lys Asp Gly Gly Ile Ser Gln Phe Ile Gly AspLys Leu Lys Pro Lys 580 585 590 act gag tat gta atc caa tat act gtt aaagga aaa cct tct att cat 1941 Thr Glu Tyr Val Ile Gln Tyr Thr Val Lys GlyLys Pro Ser Ile His 595 600 605 tta aaa gat gaa aat act gga tat att cattat gaa gat aca aat aat 1989 Leu Lys Asp Glu Asn Thr Gly Tyr Ile His TyrGlu Asp Thr Asn Asn 610 615 620 aat tta gaa gat tat caa act att aat aaacgt ttt act aca gga act 2037 Asn Leu Glu Asp Tyr Gln Thr Ile Asn Lys ArgPhe Thr Thr Gly Thr 625 630 635 640 gat tta aag gga gtg tat tta att ttaaaa agt caa aat gga gat gaa 2085 Asp Leu Lys Gly Val Tyr Leu Ile Leu LysSer Gln Asn Gly Asp Glu 645 650 655 gct tgg gga gat aac ttt att att ttggaa att agt cct tct gaa aag 2133 Ala Trp Gly Asp Asn Phe Ile Ile Leu GluIle Ser Pro Ser Glu Lys 660 665 670 tta tta agt cca gaa tta att aat acaaat aat tgg acg agt acg gga 2181 Leu Leu Ser Pro Glu Leu Ile Asn Thr AsnAsn Trp Thr Ser Thr Gly 675 680 685 tca act aat att agc ggt aat aca ctcact ctt tat cag gga gga cga 2229 Ser Thr Asn Ile Ser Gly Asn Thr Leu ThrLeu Tyr Gln Gly Gly Arg 690 695 700 ggg att cta aaa caa aac ctt caa ttagat agt ttt tca act tat aga 2277 Gly Ile Leu Lys Gln Asn Leu Gln Leu AspSer Phe Ser Thr Tyr Arg 705 710 715 720 gtg tat ttc tct gtg tcc gga gatgct aat gta agg att aga aat tct 2325 Val Tyr Phe Ser Val Ser Gly Asp AlaAsn Val Arg Ile Arg Asn Ser 725 730 735 agg gaa gtg tta ttt gaa aaa agatat atg agc ggt gct aaa gat gtt 2373 Arg Glu Val Leu Phe Glu Lys Arg TyrMet Ser Gly Ala Lys Asp Val 740 745 750 tct gaa atg ttc act aca aaa tttgag aaa gat aac ttc tat ata gag 2421 Ser Glu Met Phe Thr Thr Lys Phe GluLys Asp Asn Phe Tyr Ile Glu 755 760 765 ctt tct caa ggg aat aat tta tatggt ggt cct att gta cat ttt tac 2469 Leu Ser Gln Gly Asn Asn Leu Tyr GlyGly Pro Ile Val His Phe Tyr 770 775 780 gat gtc tct att aag taagatcgggatctaatatt aacagttttt agaagctaat 2524 Asp Val Ser Ile Lys 785 tcttgtataatgtccttgat tatggaaaaa cacaattttg tttgctaaga tgtatatata 2584 gctcactcattaaaaggcaa tcaagctt 2612 4 789 PRT Bacillus thuringiensis 4 Met Asn LysAsn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe 1 5 10 15 Ile AspTyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp 20 25 30 Ile MetAsn Met Ile Phe Lys Thr Asp Thr Gly Gly Asp Leu Thr Leu 35 40 45 Asp GluIle Leu Lys Asn Gln Gln Leu Leu Asn Asp Ile Ser Gly Lys 50 55 60 Leu AspGly Val Asn Gly Ser Leu Asn Asp Leu Ile Ala Gln Gly Asn 65 70 75 80 LeuAsn Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ala Asn Glu Gln 85 90 95 AsnGln Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile Asn Thr 100 105 110Met Leu Arg Val Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Val 115 120125 Met Lys Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu Ser Lys 130135 140 Gln Leu Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val Asn Val145 150 155 160 Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr GlnArg Ile 165 170 175 Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe AlaThr Glu Thr 180 185 190 Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Ala AspIle Arg Asp Glu 195 200 205 Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser ValThr Lys Asn Asp Val 210 215 220 Asp Gly Phe Glu Phe Tyr Leu Asn Thr PheHis Asp Val Met Val Gly 225 230 235 240 Asn Asn Leu Phe Gly Arg Ser AlaLeu Lys Thr Ala Ser Glu Leu Ile 245 250 255 Thr Lys Glu Asn Val Lys ThrSer Gly Ser Glu Val Gly Asn Val Tyr 260 265 270 Asn Phe Leu Ile Val LeuThr Ala Leu Gln Ala Lys Ala Phe Leu Thr 275 280 285 Leu Thr Pro Cys ArgLys Leu Leu Gly Leu Ala Asp Ile Asp Tyr Thr 290 295 300 Ser Ile Met AsnGlu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val 305 310 315 320 Asn IleLeu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Ala 325 330 335 LysVal Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Lys 340 345 350Pro Gly His Ala Leu Ile Gly Phe Glu Ile Ser Asn Asp Ser Ile Thr 355 360365 Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gln Asn Tyr Gln Val Asp 370375 380 Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys Leu Leu385 390 395 400 Cys Pro Asp Gln Ser Gly Gln Ile Tyr Tyr Thr Asn Asn IleVal Phe 405 410 415 Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr LysLys Met Lys 420 425 430 Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr AspSer Ser Thr Gly 435 440 445 Glu Ile Asp Leu Asn Lys Lys Lys Val Glu SerSer Glu Ala Glu Tyr 450 455 460 Arg Thr Leu Ser Ala Asn Asp Asp Gly ValTyr Met Pro Leu Gly Val 465 470 475 480 Ile Ser Glu Thr Phe Leu Thr ProIle Asn Gly Phe Gly Leu Gln Ala 485 490 495 Asp Glu Asn Ser Arg Leu IleThr Leu Thr Cys Lys Ser Tyr Leu Arg 500 505 510 Glu Leu Leu Leu Ala ThrAsp Leu Ser Asn Lys Glu Thr Lys Leu Ile 515 520 525 Val Pro Pro Ser GlyPhe Ile Ser Asn Ile Val Glu Asn Gly Ser Ile 530 535 540 Glu Glu Asp AsnLeu Glu Pro Trp Lys Ala Asn Asn Lys Asn Ala Tyr 545 550 555 560 Val AspHis Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Val His 565 570 575 LysAsp Gly Gly Ile Ser Gln Phe Ile Gly Asp Lys Leu Lys Pro Lys 580 585 590Thr Glu Tyr Val Ile Gln Tyr Thr Val Lys Gly Lys Pro Ser Ile His 595 600605 Leu Lys Asp Glu Asn Thr Gly Tyr Ile His Tyr Glu Asp Thr Asn Asn 610615 620 Asn Leu Glu Asp Tyr Gln Thr Ile Asn Lys Arg Phe Thr Thr Gly Thr625 630 635 640 Asp Leu Lys Gly Val Tyr Leu Ile Leu Lys Ser Gln Asn GlyAsp Glu 645 650 655 Ala Trp Gly Asp Asn Phe Ile Ile Leu Glu Ile Ser ProSer Glu Lys 660 665 670 Leu Leu Ser Pro Glu Leu Ile Asn Thr Asn Asn TrpThr Ser Thr Gly 675 680 685 Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr LeuTyr Gln Gly Gly Arg 690 695 700 Gly Ile Leu Lys Gln Asn Leu Gln Leu AspSer Phe Ser Thr Tyr Arg 705 710 715 720 Val Tyr Phe Ser Val Ser Gly AspAla Asn Val Arg Ile Arg Asn Ser 725 730 735 Arg Glu Val Leu Phe Glu LysArg Tyr Met Ser Gly Ala Lys Asp Val 740 745 750 Ser Glu Met Phe Thr ThrLys Phe Glu Lys Asp Asn Phe Tyr Ile Glu 755 760 765 Leu Ser Gln Gly AsnAsn Leu Tyr Gly Gly Pro Ile Val His Phe Tyr 770 775 780 Asp Val Ser IleLys 785 5 2364 DNA Bacillus thuringiensis CDS (56)..(2293) Native DNAsequence encoding VIP3A(c) from AB51 5 aatacaattt acgagggata agtgttacaaagaatagctg aggagggaga tgaac atg 58 Met 1 aac aag aat aat gct aaa tta agcaca aga gcc tta cca agt ttt att 106 Asn Lys Asn Asn Ala Lys Leu Ser ThrArg Ala Leu Pro Ser Phe Ile 5 10 15 gat tat ttc aat ggc att tat gga tttgcc act ggt atc aaa gac att 154 Asp Tyr Phe Asn Gly Ile Tyr Gly Phe AlaThr Gly Ile Lys Asp Ile 20 25 30 atg aac atg att ttt aaa acg gat aca ggtggt gat cta gcc cta gac 202 Met Asn Met Ile Phe Lys Thr Asp Thr Gly GlyAsp Leu Ala Leu Asp 35 40 45 gaa att tta gag aat cag cag cta cta aat gatatt tct ggt aaa ttg 250 Glu Ile Leu Glu Asn Gln Gln Leu Leu Asn Asp IleSer Gly Lys Leu 50 55 60 65 gat ggg gtg aat gga agc tta aat gat ctt atcgca cag gga aac tta 298 Asp Gly Val Asn Gly Ser Leu Asn Asp Leu Ile AlaGln Gly Asn Leu 70 75 80 aat aca gaa tta tct aag gaa ata tta aaa att gcaaat gaa caa aat 346 Asn Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ala AsnGlu Gln Asn 85 90 95 caa gtt tta aat gat gtt aat aac aaa ctc gat gcg ataaat acg atg 394 Gln Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile AsnThr Met 100 105 110 ctt cgg gta tat cta cct aaa att acc tct atg ttg agtgat gta atg 442 Leu Arg Val Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser AspVal Met 115 120 125 aaa caa aat tat gcg cta agt ctg caa ata gaa tac ttaagt aaa caa 490 Lys Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu SerLys Gln 130 135 140 145 ttg caa gag att tct gat aag ttg gat att att aatgta aat gta ctt 538 Leu Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn ValAsn Val Leu 150 155 160 att aac tct aca ctt act gaa att aca cct gcg tatcaa agg att aaa 586 Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr GlnArg Ile Lys 165 170 175 tat gtg aac gaa aaa ttt gag gaa tta act ttt gctaca gaa act agt 634 Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala ThrGlu Thr Ser 180 185 190 tca aaa gta aaa aag gat ggc tct cct gca gat attcgt gat gag tta 682 Ser Lys Val Lys Lys Asp Gly Ser Pro Ala Asp Ile ArgAsp Glu Leu 195 200 205 agt gag tta act gaa cta gcg aaa agt gta aca caaaat gat gtg gat 730 Ser Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Gln AsnAsp Val Asp 210 215 220 225 ggt ttt gaa ttt tac ctt aat aca ttc cac gatgta atg gta gga aat 778 Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp ValMet Val Gly Asn 230 235 240 aat tta ttc ggg cgt tca gct tta aaa act gcatcg gaa tta att act 826 Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala SerGlu Leu Ile Thr 245 250 255 aaa gaa aat gtg aaa aca agt ggc agt gag gtcgga aat gtt tat aac 874 Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val GlyAsn Val Tyr Asn 260 265 270 ttc cta att gta tta aca gct ctg caa gca caagct ttt ctt act tta 922 Phe Leu Ile Val Leu Thr Ala Leu Gln Ala Gln AlaPhe Leu Thr Leu 275 280 285 aca cca tgc cga aaa tta tta ggc tta gca gatatt gat tat act tct 970 Thr Pro Cys Arg Lys Leu Leu Gly Leu Ala Asp IleAsp Tyr Thr Ser 290 295 300 305 att atg aat gaa cat tta aat aag gaa aaagag gaa ttt aga gta aac 1018 Ile Met Asn Glu His Leu Asn Lys Glu Lys GluGlu Phe Arg Val Asn 310 315 320 atc ctc cct aca ctt tct aat act ttt tctaat cct aat tat gca aaa 1066 Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser AsnPro Asn Tyr Ala Lys 325 330 335 gtt aaa gga agt gat gaa gat gca aag atgatt gtg gaa gct aaa cca 1114 Val Lys Gly Ser Asp Glu Asp Ala Lys Met IleVal Glu Ala Lys Pro 340 345 350 gga cat gca ttg att ggg ttt gaa att agtaat gat tca att aca gta 1162 Gly His Ala Leu Ile Gly Phe Glu Ile Ser AsnAsp Ser Ile Thr Val 355 360 365 tta aaa gta tat gag gct aag cta aaa caaaat tat caa gtc gat aag 1210 Leu Lys Val Tyr Glu Ala Lys Leu Lys Gln AsnTyr Gln Val Asp Lys 370 375 380 385 gat tcc tta tcg gaa gtt att tat ggcgat atg gat aaa tta ttg tgc 1258 Asp Ser Leu Ser Glu Val Ile Tyr Gly AspMet Asp Lys Leu Leu Cys 390 395 400 cca gat caa tct gga caa atc tat tataca aat aac ata gta ttt cca 1306 Pro Asp Gln Ser Gly Gln Ile Tyr Tyr ThrAsn Asn Ile Val Phe Pro 405 410 415 aat gaa tat gta att act aaa att gatttc act aaa aaa atg aaa act 1354 Asn Glu Tyr Val Ile Thr Lys Ile Asp PheThr Lys Lys Met Lys Thr 420 425 430 tta aga tat gag gta aca gcg aat ttttat gat tct tct aca gga gaa 1402 Leu Arg Tyr Glu Val Thr Ala Asn Phe TyrAsp Ser Ser Thr Gly Glu 435 440 445 att gac tta aat aag aaa aaa gta gaatca agt gaa gcg gag tat aga 1450 Ile Asp Leu Asn Lys Lys Lys Val Glu SerSer Glu Ala Glu Tyr Arg 450 455 460 465 acg tta agt gct aat gat gat ggggtg tat atg ccg tta ggt gtc atc 1498 Thr Leu Ser Ala Asn Asp Asp Gly ValTyr Met Pro Leu Gly Val Ile 470 475 480 agt gaa aca ttt ttg act ccg attaat ggg ttt ggc ctc caa gct gat 1546 Ser Glu Thr Phe Leu Thr Pro Ile AsnGly Phe Gly Leu Gln Ala Asp 485 490 495 gaa aat tca aga tta att act ttaaca tgt aaa tca tat tta aga gaa 1594 Glu Asn Ser Arg Leu Ile Thr Leu ThrCys Lys Ser Tyr Leu Arg Glu 500 505 510 cta ctg cta gca aca gac tta agcaat aaa gaa act aaa ttg atc gtc 1642 Leu Leu Leu Ala Thr Asp Leu Ser AsnLys Glu Thr Lys Leu Ile Val 515 520 525 ccg cca agt ggt ttt att agc aatatt gta gag aac ggg tcc ata gaa 1690 Pro Pro Ser Gly Phe Ile Ser Asn IleVal Glu Asn Gly Ser Ile Glu 530 535 540 545 gag gac aat tta gag ccg tggaaa gca aat aat aag aat gcg tat gta 1738 Glu Asp Asn Leu Glu Pro Trp LysAla Asn Asn Lys Asn Ala Tyr Val 550 555 560 gat cat aca ggc gga gtg aatgga act aaa gct tta tat gtt cat aag 1786 Asp His Thr Gly Gly Val Asn GlyThr Lys Ala Leu Tyr Val His Lys 565 570 575 gac gga gga att tca caa tttatt gga gat aag tta aaa ccg aaa act 1834 Asp Gly Gly Ile Ser Gln Phe IleGly Asp Lys Leu Lys Pro Lys Thr 580 585 590 gag tat gta atc caa tat actgtt aaa gga aaa cct tct att cat tta 1882 Glu Tyr Val Ile Gln Tyr Thr ValLys Gly Lys Pro Ser Ile His Leu 595 600 605 aaa gat gaa aat act gga tatatt cat tat gaa gat aca aat aat aat 1930 Lys Asp Glu Asn Thr Gly Tyr IleHis Tyr Glu Asp Thr Asn Asn Asn 610 615 620 625 tta gaa gat tat caa actatt aat aaa cgt ttt act aca gga act gat 1978 Leu Glu Asp Tyr Gln Thr IleAsn Lys Arg Phe Thr Thr Gly Thr Asp 630 635 640 tta aag gga gtg tat ttaatt tta aaa agt caa aat gga gat gaa gct 2026 Leu Lys Gly Val Tyr Leu IleLeu Lys Ser Gln Asn Gly Asp Glu Ala 645 650 655 tgg gga gat aac ttt attatt ttg gaa att agt cct tct gaa aag tta 2074 Trp Gly Asp Asn Phe Ile IleLeu Glu Ile Ser Pro Ser Glu Lys Leu 660 665 670 tta agt cca gaa tta attaat aca aat aat tgg acg agt acg gga tca 2122 Leu Ser Pro Glu Leu Ile AsnThr Asn Asn Trp Thr Ser Thr Gly Ser 675 680 685 act aat att agc ggt aataca ctc act ctt tat cag gga gga cga ggg 2170 Thr Asn Ile Ser Gly Asn ThrLeu Thr Leu Tyr Gln Gly Gly Arg Gly 690 695 700 705 att cta aaa caa aacctt caa tta gat agt ttt tca act tat aga gtg 2218 Ile Leu Lys Gln Asn LeuGln Leu Asp Ser Phe Ser Thr Tyr Arg Val 710 715 720 tat ttc tct gtg tccgga gat gct aat gta agg att aga aat tct agg 2266 Tyr Phe Ser Val Ser GlyAsp Ala Asn Val Arg Ile Arg Asn Ser Arg 725 730 735 gaa gtg tta ttt gaaaaa aag gat ata tgagcggcgc taaagatgtt 2313 Glu Val Leu Phe Glu Lys LysAsp Ile 740 745 tctgaaatgt tcactacaaa attgaaagat aacttctata tagagctttc t2364 6 746 PRT Bacillus thuringiensis 6 Met Asn Lys Asn Asn Ala Lys LeuSer Thr Arg Ala Leu Pro Ser Phe 1 5 10 15 Ile Asp Tyr Phe Asn Gly IleTyr Gly Phe Ala Thr Gly Ile Lys Asp 20 25 30 Ile Met Asn Met Ile Phe LysThr Asp Thr Gly Gly Asp Leu Ala Leu 35 40 45 Asp Glu Ile Leu Glu Asn GlnGln Leu Leu Asn Asp Ile Ser Gly Lys 50 55 60 Leu Asp Gly Val Asn Gly SerLeu Asn Asp Leu Ile Ala Gln Gly Asn 65 70 75 80 Leu Asn Thr Glu Leu SerLys Glu Ile Leu Lys Ile Ala Asn Glu Gln 85 90 95 Asn Gln Val Leu Asn AspVal Asn Asn Lys Leu Asp Ala Ile Asn Thr 100 105 110 Met Leu Arg Val TyrLeu Pro Lys Ile Thr Ser Met Leu Ser Asp Val 115 120 125 Met Lys Gln AsnTyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu Ser Lys 130 135 140 Gln Leu GlnGlu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val Asn Val 145 150 155 160 LeuIle Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Ile 165 170 175Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr 180 185190 Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Ala Asp Ile Arg Asp Glu 195200 205 Leu Ser Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Gln Asn Asp Val210 215 220 Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met ValGly 225 230 235 240 Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala SerGlu Leu Ile 245 250 255 Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu ValGly Asn Val Tyr 260 265 270 Asn Phe Leu Ile Val Leu Thr Ala Leu Gln AlaGln Ala Phe Leu Thr 275 280 285 Leu Thr Pro Cys Arg Lys Leu Leu Gly LeuAla Asp Ile Asp Tyr Thr 290 295 300 Ser Ile Met Asn Glu His Leu Asn LysGlu Lys Glu Glu Phe Arg Val 305 310 315 320 Asn Ile Leu Pro Thr Leu SerAsn Thr Phe Ser Asn Pro Asn Tyr Ala 325 330 335 Lys Val Lys Gly Ser AspGlu Asp Ala Lys Met Ile Val Glu Ala Lys 340 345 350 Pro Gly His Ala LeuIle Gly Phe Glu Ile Ser Asn Asp Ser Ile Thr 355 360 365 Val Leu Lys ValTyr Glu Ala Lys Leu Lys Gln Asn Tyr Gln Val Asp 370 375 380 Lys Asp SerLeu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys Leu Leu 385 390 395 400 CysPro Asp Gln Ser Gly Gln Ile Tyr Tyr Thr Asn Asn Ile Val Phe 405 410 415Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Lys 420 425430 Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly 435440 445 Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Tyr450 455 460 Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu GlyVal 465 470 475 480 Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe GlyLeu Gln Ala 485 490 495 Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys LysSer Tyr Leu Arg 500 505 510 Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn LysGlu Thr Lys Leu Ile 515 520 525 Val Pro Pro Ser Gly Phe Ile Ser Asn IleVal Glu Asn Gly Ser Ile 530 535 540 Glu Glu Asp Asn Leu Glu Pro Trp LysAla Asn Asn Lys Asn Ala Tyr 545 550 555 560 Val Asp His Thr Gly Gly ValAsn Gly Thr Lys Ala Leu Tyr Val His 565 570 575 Lys Asp Gly Gly Ile SerGln Phe Ile Gly Asp Lys Leu Lys Pro Lys 580 585 590 Thr Glu Tyr Val IleGln Tyr Thr Val Lys Gly Lys Pro Ser Ile His 595 600 605 Leu Lys Asp GluAsn Thr Gly Tyr Ile His Tyr Glu Asp Thr Asn Asn 610 615 620 Asn Leu GluAsp Tyr Gln Thr Ile Asn Lys Arg Phe Thr Thr Gly Thr 625 630 635 640 AspLeu Lys Gly Val Tyr Leu Ile Leu Lys Ser Gln Asn Gly Asp Glu 645 650 655Ala Trp Gly Asp Asn Phe Ile Ile Leu Glu Ile Ser Pro Ser Glu Lys 660 665670 Leu Leu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gly 675680 685 Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gln Gly Gly Arg690 695 700 Gly Ile Leu Lys Gln Asn Leu Gln Leu Asp Ser Phe Ser Thr TyrArg 705 710 715 720 Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg IleArg Asn Ser 725 730 735 Arg Glu Val Leu Phe Glu Lys Lys Asp Ile 740 7457 2403 DNA Artificial Sequence Description of Artificial Sequence maizeoptimized sequence encoding VIP3A(a) 7 ggatccacca atgaacatga acaagaacaacaccaagctg agcacccgcg ccctgccgag 60 cttcatcgac tacttcaacg gcatctacggcttcgccacc ggcatcaagg acatcatgaa 120 catgatcttc aagaccgaca ccggcggcgacctgaccctg gacgagatcc tgaagaacca 180 gcagctgctg aacgacatca gcggcaagctggacggcgtg aacggcagcc tgaacgacct 240 gatcgcccag ggcaacctga acaccgagctgagcaaggag atccttaaga tcgccaacga 300 gcagaaccag gtgctgaacg acgtgaacaacaagctggac gccatcaaca ccatgctgcg 360 cgtgtacctg ccgaagatca ccagcatgctgagcgacgtg atgaagcaga actacgccct 420 gagcctgcag atcgagtacc tgagcaagcagctgcaggag atcagcgaca agctggacat 480 catcaacgtg aacgtcctga tcaacagcaccctgaccgag atcaccccgg cctaccagcg 540 catcaagtac gtgaacgaga agttcgaagagctgaccttc gccaccgaga ccagcagcaa 600 ggtgaagaag gacggcagcc cggccgacatcctggacgag ctgaccgagc tgaccgagct 660 ggccaagagc gtgaccaaga acgacgtggacggcttcgag ttctacctga acaccttcca 720 cgacgtgatg gtgggcaaca acctgttcggccgcagcgcc ctgaagaccg ccagcgagct 780 gatcaccaag gagaacgtga agaccagcggcagcgaggtg ggcaacgtgt acaacttcct 840 gatcgtgctg accgccctgc aggcccaggccttcctgacc ctgaccacct gtcgcaagct 900 gctgggcctg gccgacatcg actacaccagcatcatgaac gagcacttga acaaggagaa 960 ggaggagttc cgcgtgaaca tcctgccgaccctgagcaac accttcagca acccgaacta 1020 cgccaaggtg aagggcagcg acgaggacgccaagatgatc gtggaggcta agccgggcca 1080 cgcgttgatc ggcttcgaga tcagcaacgacagcatcacc gtgctgaagg tgtacgaggc 1140 caagctgaag cagaactacc aggtggacaaggacagcttg agcgaggtga tctacggcga 1200 catggacaag ctgctgtgtc cggaccagagcgagcaaatc tactacacca acaacatcgt 1260 gttcccgaac gagtacgtga tcaccaagatcgacttcacc aagaagatga agaccctgcg 1320 ctacgaggtg accgccaact tctacgacagcagcaccggc gagatcgacc tgaacaagaa 1380 gaaggtggag agcagcgagg ccgagtaccgcaccctgagc gcgaacgacg acggcgtcta 1440 catgccactg ggcgtgatca gcgagaccttcctgaccccg atcaacggct ttggcctgca 1500 ggccgacgag aacagccgcc tgatcaccctgacctgtaag agctacctgc gcgagctgct 1560 gctagccacc gacctgagca acaaggagaccaagctgatc gtgccaccga gcggcttcat 1620 cagcaacatc gtggagaacg gcagcatcgaggaggacaac ctggagccgt ggaaggccaa 1680 caacaagaac gcctacgtgg accacaccggcggcgtgaac ggcaccaagg ccctgtacgt 1740 gcacaaggac ggcggcatca gccagttcatcggcgacaag ctgaagccga agaccgagta 1800 cgtgatccag tacaccgtga agggcaagccatcgattcac ctgaaggacg agaacaccgg 1860 ctacatccac tacgaggaca ccaacaacaacctggaggac taccagacca tcaacaagcg 1920 cttcaccacc ggcaccgacc tgaagggcgtgtacctgatc ctgaagagcc agaacggcga 1980 cgaggcctgg ggcgacaact tcatcatcctggagatcagc ccgagcgaga agctgctgag 2040 cccggagctg atcaacacca acaactggaccagcaccggc agcaccaaca tcagcggcaa 2100 caccctgacc ctgtaccagg gcggccgcggcatcctgaag cagaacctgc agctggacag 2160 cttcagcacc taccgcgtgt acttcagcgtgagcggcgac gccaacgtgc gcatccgcaa 2220 cagccgcgag gtgctgttcg agaagaggtacatgagcggc gccaaggacg tgagcgagat 2280 gttcaccacc aagttcgaga aggacaacttctacatcgag ctgagccagg gcaacaacct 2340 gtacggcggc ccgatcgtgc acttctacgacgtgagcatc aagttaacgt agagctcaga 2400 tct 2403 8 1638 DNA Agrotisipsilon CDS (2)..(1189) Translation of cDNA encoding VIP3A(a) receptorfrom Black cutworm 8 t agt gga tcc ccc ggg ctg cag gaa ttc gcg gcc gcgtcg acc atg tac 49 Ser Gly Ser Pro Gly Leu Gln Glu Phe Ala Ala Ala SerThr Met Tyr 1 5 10 15 tct aga ata ttt ttc ctc ctt gtg ata gtg tgt gctgtt aag gct tct 97 Ser Arg Ile Phe Phe Leu Leu Val Ile Val Cys Ala ValLys Ala Ser 20 25 30 ctg ttt act gta aat gtg tat gat gat aac ccc gaa actgaa att gcg 145 Leu Phe Thr Val Asn Val Tyr Asp Asp Asn Pro Glu Thr GluIle Ala 35 40 45 agt agt cta aaa ggc tgt aac ccc caa gag tgt gac cag cggtgt cgt 193 Ser Ser Leu Lys Gly Cys Asn Pro Gln Glu Cys Asp Gln Arg CysArg 50 55 60 aga ctg aag ttt ccc ggt ggc gcc tgt gtc aat ggt cgc tgc aagtgt 241 Arg Leu Lys Phe Pro Gly Gly Ala Cys Val Asn Gly Arg Cys Lys Cys65 70 75 80 gac aac ttc ctc agt gta aaa gat gac gtg tct gtt gaa gag cctgcg 289 Asp Asn Phe Leu Ser Val Lys Asp Asp Val Ser Val Glu Glu Pro Ala85 90 95 att ctc aaa gat ttg gtg tca tta gaa gct gaa cag gca gcg aaa agt337 Ile Leu Lys Asp Leu Val Ser Leu Glu Ala Glu Gln Ala Ala Lys Ser 100105 110 aga tgc aga aac aga gtg tgt gac gcg gtg tgc cgt gcc cta cac aac385 Arg Cys Arg Asn Arg Val Cys Asp Ala Val Cys Arg Ala Leu His Asn 115120 125 acc agt ggt gcc tgt gtt gat gga caa tgc aag tgt act aat aag atc433 Thr Ser Gly Ala Cys Val Asp Gly Gln Cys Lys Cys Thr Asn Lys Ile 130135 140 agt gca gga gat att gtg tct gat cct gct gaa tcg cta cgc act tgt481 Ser Ala Gly Asp Ile Val Ser Asp Pro Ala Glu Ser Leu Arg Thr Cys 145150 155 160 aac cct ata agg tgt gac gaa caa tgt aga aga aat ggc cat gaattt 529 Asn Pro Ile Arg Cys Asp Glu Gln Cys Arg Arg Asn Gly His Glu Phe165 170 175 ggt gtt tgc ttc aaa gga caa tgc aag tgt gat tac ttc ctc aaggaa 577 Gly Val Cys Phe Lys Gly Gln Cys Lys Cys Asp Tyr Phe Leu Lys Glu180 185 190 gaa gtc gat gaa cct gaa gtt aca agc ctt cca aaa aac tgc aacccc 625 Glu Val Asp Glu Pro Glu Val Thr Ser Leu Pro Lys Asn Cys Asn Pro195 200 205 caa gag tgt gac cag cgt tgt cgt aga ctg aag ttc ccc ggt ggcgcc 673 Gln Glu Cys Asp Gln Arg Cys Arg Arg Leu Lys Phe Pro Gly Gly Ala210 215 220 tgt gtc aac ggg cgc tgc aag tgt gac aac ttc ttc agt gca ggagat 721 Cys Val Asn Gly Arg Cys Lys Cys Asp Asn Phe Phe Ser Ala Gly Asp225 230 235 240 att gtg tct gat cct gcc gaa tcg cta cgc tct tgt aac cctata agg 769 Ile Val Ser Asp Pro Ala Glu Ser Leu Arg Ser Cys Asn Pro IleArg 245 250 255 tgt gac gaa caa tgt aga aga aat ggc cat gaa ttt ggt gtttgc ttc 817 Cys Asp Glu Gln Cys Arg Arg Asn Gly His Glu Phe Gly Val CysPhe 260 265 270 aaa gga caa tgc aag tgt gat tac ttc ctc aac tca gaa gtagac gct 865 Lys Gly Gln Cys Lys Cys Asp Tyr Phe Leu Asn Ser Glu Val AspAla 275 280 285 gtt aat gag ttt cct caa gcg ggc tca aaa cgc tac tgc aactta acg 913 Val Asn Glu Phe Pro Gln Ala Gly Ser Lys Arg Tyr Cys Asn LeuThr 290 295 300 caa tgc aac cag acg tgc gcc aat cgt ttc tat gat agt gctaga gtg 961 Gln Cys Asn Gln Thr Cys Ala Asn Arg Phe Tyr Asp Ser Ala ArgVal 305 310 315 320 atc cac ggc tgg tgc aaa tgc tac agt aag atg gaa agacag gat gca 1009 Ile His Gly Trp Cys Lys Cys Tyr Ser Lys Met Glu Arg GlnAsp Ala 325 330 335 tct cca tta aac gat gtg act gag gat gaa aat gaa gtttct aac gat 1057 Ser Pro Leu Asn Asp Val Thr Glu Asp Glu Asn Glu Val SerAsn Asp 340 345 350 atc ctg agg act gtt gca gag gag ctg tct gat gtg tcacct agg gcc 1105 Ile Leu Arg Thr Val Ala Glu Glu Leu Ser Asp Val Ser ProArg Ala 355 360 365 tgc aaa tca gcg agc tgc aat caa gca tgt cgc gcc ttctac ttt aaa 1153 Cys Lys Ser Ala Ser Cys Asn Gln Ala Cys Arg Ala Phe TyrPhe Lys 370 375 380 gga ggg tgg tgt cgc ttt gga cga tgc caa tgc ttctaaaattagt 1199 Gly Gly Trp Cys Arg Phe Gly Arg Cys Gln Cys Phe 385 390395 atgatatatg aattttgtat tattcggtta attgtgttat gtttaaaaaa cataatgtct1259 tcattttaga aaaaagtacc ttcactaaag cgcaacaatt aactagtagt taattattaa1319 ctagtagtta aattattgat gattatgatt atcttagtag tagttaatta taatcatcaa1379 ctattaacta gtagttaatt attaactagt agttaaatta ttgatgatta tgattatctt1439 agtagtagtt aattattgtt tcttataata atctagtatg ttggtaggta cttaataata1499 acgcttctga caaaaaattt aaaattaaat aattctatca aacataaata ataactgaaa1559 taaaaattta taagagaaaa aaaaaaagtc gacgcggccg cgaattcgat atcaagctta1619 tcgataccgt cgacctcga 1638 9 396 PRT Agrotis ipsilon 9 Ser Gly SerPro Gly Leu Gln Glu Phe Ala Ala Ala Ser Thr Met Tyr 1 5 10 15 Ser ArgIle Phe Phe Leu Leu Val Ile Val Cys Ala Val Lys Ala Ser 20 25 30 Leu PheThr Val Asn Val Tyr Asp Asp Asn Pro Glu Thr Glu Ile Ala 35 40 45 Ser SerLeu Lys Gly Cys Asn Pro Gln Glu Cys Asp Gln Arg Cys Arg 50 55 60 Arg LeuLys Phe Pro Gly Gly Ala Cys Val Asn Gly Arg Cys Lys Cys 65 70 75 80 AspAsn Phe Leu Ser Val Lys Asp Asp Val Ser Val Glu Glu Pro Ala 85 90 95 IleLeu Lys Asp Leu Val Ser Leu Glu Ala Glu Gln Ala Ala Lys Ser 100 105 110Arg Cys Arg Asn Arg Val Cys Asp Ala Val Cys Arg Ala Leu His Asn 115 120125 Thr Ser Gly Ala Cys Val Asp Gly Gln Cys Lys Cys Thr Asn Lys Ile 130135 140 Ser Ala Gly Asp Ile Val Ser Asp Pro Ala Glu Ser Leu Arg Thr Cys145 150 155 160 Asn Pro Ile Arg Cys Asp Glu Gln Cys Arg Arg Asn Gly HisGlu Phe 165 170 175 Gly Val Cys Phe Lys Gly Gln Cys Lys Cys Asp Tyr PheLeu Lys Glu 180 185 190 Glu Val Asp Glu Pro Glu Val Thr Ser Leu Pro LysAsn Cys Asn Pro 195 200 205 Gln Glu Cys Asp Gln Arg Cys Arg Arg Leu LysPhe Pro Gly Gly Ala 210 215 220 Cys Val Asn Gly Arg Cys Lys Cys Asp AsnPhe Phe Ser Ala Gly Asp 225 230 235 240 Ile Val Ser Asp Pro Ala Glu SerLeu Arg Ser Cys Asn Pro Ile Arg 245 250 255 Cys Asp Glu Gln Cys Arg ArgAsn Gly His Glu Phe Gly Val Cys Phe 260 265 270 Lys Gly Gln Cys Lys CysAsp Tyr Phe Leu Asn Ser Glu Val Asp Ala 275 280 285 Val Asn Glu Phe ProGln Ala Gly Ser Lys Arg Tyr Cys Asn Leu Thr 290 295 300 Gln Cys Asn GlnThr Cys Ala Asn Arg Phe Tyr Asp Ser Ala Arg Val 305 310 315 320 Ile HisGly Trp Cys Lys Cys Tyr Ser Lys Met Glu Arg Gln Asp Ala 325 330 335 SerPro Leu Asn Asp Val Thr Glu Asp Glu Asn Glu Val Ser Asn Asp 340 345 350Ile Leu Arg Thr Val Ala Glu Glu Leu Ser Asp Val Ser Pro Arg Ala 355 360365 Cys Lys Ser Ala Ser Cys Asn Gln Ala Cys Arg Ala Phe Tyr Phe Lys 370375 380 Gly Gly Trp Cys Arg Phe Gly Arg Cys Gln Cys Phe 385 390 395 1014 PRT Bacillus thuringiensis N-terminal amino acid sequence of proteinknown as anion exchange fraction 23 (smaller) 10 Xaa Glu Pro Phe Val SerAla Xaa Xaa Xaa Gln Xaa Xaa Xaa 1 5 10 11 13 PRT Bacillus thuringiensisSITE (1)..(13) Xaa represents any amino acid 11 Xaa Glu Tyr Glu Asn ValGlu Pro Phe Val Ser Ala Xaa 1 5 10 12 14 PRT Bacillus thuringiensis 12Met Asn Lys Asn Asn Thr Lys Leu Pro Thr Arg Ala Leu Pro 1 5 10 13 15 PRTBacillus thuringiensis 13 Ala Leu Ser Glu Asn Thr Gly Lys Asp Gly GlyTyr Ile Val Pro 1 5 10 15 14 9 PRT Bacillus thuringiensis 14 Met Asp AsnAsn Pro Asn Ile Asn Glu 1 5 15 9 PRT Bacillus thuringiensis 15 Met AspAsn Asn Pro Asn Ile Asn Glu 1 5 16 11 PRT Bacillus thuringiensis 16 MetAsn Val Leu Asn Ser Gly Arg Thr Thr Ile 1 5 10 17 17 DNA ArtificialSequence Description of Artificial Sequence primer sequence 17cgattaatgt tggcctc 17 18 21 DNA Artificial Sequence Description ofArtificial Sequence primer sequence 18 cattagcatc tccggacaca g 21 192370 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA encoding VIP3A(b) 19 atgaacaaga acaacaccaa gctgagcacccgcgccctgc cgagcttcat cgactacttc 60 aacggcatct acggcttcgc caccggcatcaaggacatca tgaacatgat cttcaagacc 120 gacaccggcg gcgacctgac cctggacgagatcctgaaga accagcagct gctgaacgac 180 atcagcggca agctggacgg cgtgaacggcagcctgaacg acctgatcgc ccagggcaac 240 ctgaacaccg agctgagcaa ggagatccttaagatcgcca acgagcagaa ccaggtgctg 300 aacgacgtga acaacaagct ggacgccatcaacaccatgc tgcgcgtgta cctgccgaag 360 atcaccagca tgctgagcga cgtgatgaagcagaactacg ccctgagcct gcagatcgag 420 tacctgagca agcagctgca ggagatcagcgacaagctgg acatcatcaa cgtgaacgtc 480 ctgatcaaca gcaccctgac cgagatcaccccggcctacc agcgcatcaa gtacgtgaac 540 gagaagttcg aagagctgac cttcgccaccgagaccagca gcaaggtgaa gaaggacggc 600 agcccggccg acatcctgga cgagctgaccgagctgaccg agctggcgaa gagcgtgacc 660 aagaacgacg tggacggctt cgagttctacctgaacacct tccacgacgt gatggtgggc 720 aacaacctgt tcggccgcag cgccctgaagaccgccagcg agctgatcac caaggagaac 780 gtgaagacca gcggcagcga ggtgggcaacgtgtacaact tcctgatcgt gctgaccgcc 840 ctgcaggcca aggccttcct gaccctgaccccctgtcgca agctgctggg cctggccgac 900 atcgactaca ccagcatcat gaacgagcacttgaacaagg agaaggagga gttccgcgtg 960 aacatcctgc cgaccctgag caacaccttcagcaacccga actacgccaa ggtgaagggc 1020 agcgacgagg acgccaagat gatcgtggaggctaagccgg gccacgcgtt gatcggcttc 1080 gagatcagca acgacagcat caccgtgctgaaggtgtacg aggccaagct gaagcagaac 1140 taccaggtgg acaaggacag cttgagcgaggtgatctacg gcgacatgga caagctgctg 1200 tgtccggacc agagcgggca aatctactacaccaacaaca tcgtgttccc gaacgagtac 1260 gtgatcacca agatcgactt caccaagaagatgaagaccc tgcgctacga ggtgaccgcc 1320 aacttctacg acagcagcac cggcgagatcgacctgaaca agaagaaggt ggagagcagc 1380 gaggccgagt accgcaccct gagcgcgaacgacgacggcg tctacatgcc actgggcgtg 1440 atcagcgaga ccttcctgac cccgatcaacggctttggcc tgcaggccga cgagaacagc 1500 cgcctgatca ccctgacctg taagagctacctgcgcgagc tgctgctagc caccgacctg 1560 agcaacaagg agaccaagct gatcgtgccaccgagcggct tcatcagcaa catcgtggag 1620 aacggcagca tcgaggagga caacctggagccgtggaagg ccaacaacaa gaacgcctac 1680 gtggaccaca ccggcggcgt gaacggcaccaaggccctgt acgtgcacaa ggacggcggc 1740 atcagccagt tcatcggcga caagctgaagccgaagaccg agtacgtgat ccagtacacc 1800 gtgaagggca agccatcgat tcacctgaaggacgagaaca ccggctacat ccactacgag 1860 gacaccaaca acaacctgga ggactaccagaccatcaaca agcgcttcac caccggcacc 1920 gacctgaagg gcgtgtacct gatcctgaagagccagaacg gcgacgaggc ctggggcgac 1980 aacttcatca tcctggagat cagcccgagcgagaagctgc tgagcccgga gctgatcaac 2040 accaacaact ggaccagcac cggcagcaccaacatcagcg gcaacaccct gaccctgtac 2100 cagggcggcc gcggcatcct gaagcagaacctgcagctgg acagcttcag cacctaccgc 2160 gtgtacttca gcgtgagcgg cgacgccaacgtgcgcatcc gcaactcccg cgaggtgctg 2220 ttcaagaaga ggtacatgag cggcgccaaggacgtgagcg agatgttcac caccaagttc 2280 gagaaggaca acttctacat cgagctgagccagggcaaca acctgtacgg cggcccgatc 2340 gtgcacttct acgacgtgag catcaagtag2370 20 2241 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA encoding VIP3A(c) 20 atgaacaaga acaacgccaa gctgagcacccgcgccctgc cgagcttcat cgactacttc 60 aacggcatct acggcttcgc caccggcatcaaggacatca tgaacatgat cttcaagacc 120 gacaccggcg gcgacctggc cctggacgagatcctggaga accagcagct gctgaacgac 180 atcagcggca agctggacgg cgtgaacggcagcctgaacg acctgatcgc ccagggcaac 240 ctgaacaccg agctgagcaa ggagatccttaagatcgcca acgagcagaa ccaggtgctg 300 aacgacgtga acaacaagct ggacgccatcaacaccatgc tgcgcgtgta cctgccgaag 360 atcaccagca tgctgagcga cgtgatgaagcagaactacg ccctgagcct gcagatcgag 420 tacctgagca agcagctgca ggagatcagcgacaagctgg acatcatcaa cgtgaacgtc 480 ctgatcaaca gcaccctgac cgagatcaccccggcctacc agcgcatcaa gtacgtgaac 540 gagaagttcg aagagctgac cttcgccaccgagaccagca gcaaggtgaa gaaggacggc 600 agcccggccg acatccggga cgagctgagcgagctgaccg agctggcgaa gagcgtgacc 660 cagaacgacg tggacggctt cgagttctacctgaacacct tccacgacgt gatggtgggc 720 aacaacctgt tcggccgcag cgccctgaagaccgccagcg agctgatcac caaggagaac 780 gtgaagacca gcggcagcga ggtgggcaacgtgtacaact tcctgatcgt gctgaccgcc 840 ctgcaggccc aggccttcct gaccctgaccccctgtcgca agctgctggg cctggccgac 900 atcgactaca ccagcatcat gaacgagcacttgaacaagg agaaggagga gttccgcgtg 960 aacatcctgc cgaccctgag caacaccttcagcaacccga actacgccaa ggtgaagggc 1020 agcgacgagg acgccaagat gatcgtggaggctaagccgg gccacgcgtt gatcggcttc 1080 gagatcagca acgacagcat caccgtgctgaaggtgtacg aggccaagct gaagcagaac 1140 taccaggtgg acaaggacag cttgagcgaggtgatctacg gcgacatgga caagctgctg 1200 tgtccggacc agagcgggca aatctactacaccaacaaca tcgtgttccc gaacgagtac 1260 gtgatcacca agatcgactt caccaagaagatgaagaccc tgcgctacga ggtgaccgcc 1320 aacttctacg acagcagcac cggcgagatcgacctgaaca agaagaaggt ggagagcagc 1380 gaggccgagt accgcaccct gagcgcgaacgacgacggcg tctacatgcc actgggcgtg 1440 atcagcgaga ccttcctgac cccgatcaacggctttggcc tgcaggccga cgagaacagc 1500 cgcctgatca ccctgacctg taagagctacctgcgcgagc tgctgctagc caccgacctg 1560 agcaacaagg agaccaagct gatcgtgccaccgagcggct tcatcagcaa catcgtggag 1620 aacggcagca tcgaggagga caacctggagccgtggaagg ccaacaacaa gaacgcctac 1680 gtggaccaca ccggcggcgt gaacggcaccaaggccctgt acgtgcacaa ggacggcggc 1740 atcagccagt tcatcggcga caagctgaagccgaagaccg agtacgtgat ccagtacacc 1800 gtgaagggca agccatcgat tcacctgaaggacgagaaca ccggctacat ccactacgag 1860 gacaccaaca acaacctgga ggactaccagaccatcaaca agcgcttcac caccggcacc 1920 gacctgaagg gcgtgtacct gatcctgaagagccagaacg gcgacgaggc ctggggcgac 1980 aacttcatca tcctggagat cagcccgagcgagaagctgc tgagcccgga gctgatcaac 2040 accaacaact ggaccagcac cggcagcaccaacatcagcg gcaacaccct gaccctgtac 2100 cagggcggcc gcggcatcct gaagcagaacctgcagctgg acagcttcag cacctaccgc 2160 gtgtacttca gcgtgagcgg cgacgccaacgtgcgcatcc gcaactcccg cgaggtgctg 2220 ttcgagaaga aggacaagta g 2241 21 13DNA Artificial Sequence Description of Artificial Sequence Clontechsequence 21 gtcgaccatg gtc 13 22 12 DNA Artificial Sequence Descriptionof Artificial Sequence Joshi sequence 22 taaacaatgg ct 12

What is claimed is:
 1. An isolated receptor to a VIP3A(a) protein of SEQID NO:2, wherein said receptor is isolated from the midgut of blackcutworm larvae.
 2. An isolated DNA molecule comprising a sequence whichencodes a receptor according to claim
 1. 3. The receptor of claim 1,wherein said receptor comprises the amino acid sequence set forth in SEQID NO:9.
 4. The DNA molecule according to claim 2, comprising the codingsequence set forth in SEQ ID: NO:8.
 5. A method of identifying acompound as a VIP3 receptor chemical ligand having pesticidal activity,comprising exposing the receptor of claim 1 to test compound, andassaying the interaction between the receptor and the test compound. 6.The method according to claim 5, wherein the receptor is cellularlyexpressed and the assayed interaction is programmed cell death.
 7. Themethod according to claim 5, wherein the assayed interaction is specificbinding between the receptor and the test compound.
 8. A method ofidentifying a compound as a VIP3 receptor chemical ligand havingpesticidal activity, comprising exposing the receptor of claim 3 to atest compound and assaying the interaction between the receptor and thetest compound.
 9. The method according to claim 8, wherein the receptoris cellularly expressed and the assayed interaction is programmed celldeath.
 10. The method according to claim 8, wherein the assayedinteraction is specific binding between the receptor and the testcompound.